Carrier particle-drug conjugates, self-immolative linkers, and uses thereof

ABSTRACT

The disclosure relates to carrier particle-drug conjugates, including nanoparticle drug conjugates (NDC), that can be used in the delivery of a drug to a biological target (e.g., for targeted delivery of a cytotoxic drug to a cancer cell or tumor). Also disclosed are self-immolative linkers and linker-payload conjugates suitable for use in a carrier particle drug conjugate, and methods of making the same, and methods for treating cancer.

RELATED APPLICATIONS

This application filed under 35 U.S.C. 111(a) is a continuation ofInternational Application No. PCT/US2021/056621, filed on Oct. 26, 2021,which claims the benefit of U.S. Provisional Application No. 63/105,995,filed on Oct. 27, 2020, U.S. Provisional Application No. 63/116,393,filed on Nov. 20, 2020, U.S. Provisional Application No. 63/117,110,filed on Nov. 23, 2020, U.S. Provisional Application No. 63/155,043,filed on Mar. 1, 2021, U.S. Provisional Application No. 63/222,181,filed on Jul. 15, 2021, U.S. Provisional Application No. 63/242,201,filed on Sep. 9, 2021, and U.S. Provisional Application No. 63/254,837,filed on Oct. 12, 2021, the contents of which are each incorporatedherein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML file, created on Sep. 13, 2022, isnamed 761825_290010_SL.xml and is 9,091 bytes in size.

BACKGROUND OF THE INVENTION

Targeted delivery of therapeutics (e.g., cytotoxic drugs) to cancercells is an emerging approach for cancer treatment. The toxicity of thedelivered therapeutics to healthy tissue or organs in a subject can begreatly reduced by the selective delivery of drugs to a targeted diseasearea, leading to improved therapeutic outcomes. Antibody-drug-conjugates(ADCs) are a popular platform for targeted drug delivery, whichtypically feature a highly toxic drug substance covalently attached to amonoclonal antibody that can target cancer, wherein the toxic drugsubstance is released upon targeting of the cancer. However, manychallenges remain with conventional targeted drug delivery platforms,such as ADCs, including difficulties in production, limitations in drugloading capacity, poor tumor penetration, and lack of ability toovercome tumor heterogeneity.

Cornell University and Memorial Sloan Kettering Cancer Center developedultrasmall sub-10 nm silica-organic hybrid nanoparticles, referred to asCornell prime dots (C′Dots), which have significant potential indiagnostics and therapeutic applications. For example, C′Dots can beconjugated with epidermal growth factor receptor inhibitors, e.g.,gefitinib, which is a cancer-targeted agent that inhibits cancer growth(WO 2015/183882 A1). However, the mechanism of action (MOA) of EGFRinhibitors requires active binding to the epidermal growth factorreceptor, so a continuous high concentration of the payload in thetargeted cancer cell is required to effectively inhibit cancer cellproliferation. This type of MOA is generally not compatible with therelatively short blood circulation half-life of C′Dots.

Folate receptor alpha (FRα), also known as FOLR1, has receivedsignificant attention from the scientific community as a potentialtarget for cancer therapy, and other isoforms of FR have also beenidentified as potential biological targets. See, e.g., Targeting FolateReceptor Alpha For Cancer Treatment, Cheung, A., et al. Oncotarget(2016) 7 (32):52553; Targeting the folate receptor: diagnostic andtherapeutic approaches to personalize cancer treatments, Ledermann, J.A. et al., Annals of Oncology (2015), 26:2034-2043; each of which areincorporated herein by reference in their entireties. Folate receptor isrecognized as a target for certain cancers, as folate receptor can beoverexpressed in tumors, such as those of the ovary, endometrium,breast, colon, and lung, but its distribution in normal tissues is lowand restricted. Emerging insights have suggested that FR may alsoexhibit cell-growth regulation and signaling functions, in addition toserving as a folate receptor and transporter. These features togetherrender FR an attractive therapeutic target.

Folic Acid is transported into the cells by various mechanisms, and themost prevalent mechanism is mediation through Folate Receptors, of whichthere are four glycopeptide members (FR alpha [FOLR1], FR beta [FOLR2],FR gamma [FOLR3], and FR delta [FOLR4]). Among these four members, thealpha isoform (FR alpha or FRα) is a glycosylphosphatidylinositol(GPI)-anchored membrane protein with high affinity for binding andtransporting the active form of folate, 5-methyltetrahydro folate(5MTF). The alpha isoform has been reported to be over-expressed incertain solid tumors, for example, in ovarian cancer, fallopian tubecancer, primary peritoneum cancer, uterus cancer, kidney cancer, lungcancer, brain cancer, gastrointestinal cancer, and breast carcinomas.The alpha isoform is also over-expressed in certain hematologicalmalignancies, which can be exploited for treatment of thesemalignancies, e.g., for treatment of acute myeloid lymphoma (AML),including pediatric AML. This low and restricted distribution in normaltissues or cells, alongside emerging insights into tumor-promotingfunctions and association of expression with patient prognosis, togetherrender FRα an attractive therapeutic target. Additionally, the betaisoform (FRO) is overexpressed in certain cancers, e.g., hematologicalmalignancies such as acute myeloid leukemia (AML) and chronicmyelogenous leukemia (CML), providing the opportunity to developtargeted therapies for these cancers.

Although many FR-targeted drug delivery platforms have been developedand tested for cancer treatment in the past, e.g., using both ADCs andsmall-molecule drug conjugates, none of them are successfully approvedfor clinical use due to their limited therapeutic outcome (EP 0624377A2, U.S. Pat. No. 9,192,682 B2, Leamon, et al., “Comparative preclinicalactivity of the folate-targeted Vinca alkaloid conjugates EC140 andEC145, Int. J. Cancer: 121, 1585-1592 (2007), Leamon et al.,“Folate-Vinca Alkaloid Conjugates for Cancer Therapy: AStructure-Activity Relationships, Bioconjugate Chemistry, 2014, 25,560-568; Scaranti, M., et al. Exploiting the folate receptor a inoncology. Nat Rev C/in Oncol. (2020) 17: 349-359).

Therefore, successful development of additional drug-linkertechnologies, including additional FR-targeted drug delivery platforms,remain highly desired.

SUMMARY OF THE INVENTION

The present disclosure provides carrier particle drug conjugates usefulin, for example, the delivery of a drug to a biological target (e.g.,targeted delivery to a cancer cell or tumor), as well as self-immolativelinkers and linker-payload conjugates suitable for use in a carrierparticle drug conjugate, and methods of making the same, and theirmethods of use (e.g., in treating cancer).

In one aspect, the present disclosure provides a nanoparticle drugconjugate (NDC) comprising: (a) a nanoparticle that comprises asilica-based core and a silica shell surrounding at least a portion ofthe core; polyethylene glycol (PEG) covalently bonded to the surface ofthe nanoparticle, and a fluorescent compound covalently encapsulatedwithin the core of the nanoparticle; (b) a targeting ligand that bindsto folate receptor (FR), wherein the targeting ligand may be selectedfrom the group consisting of folic acid, dihydrofolic acid,tetrahydrofolic acid, and folate receptor binding derivatives of any ofthe foregoing, and wherein the targeting ligand is attached to thenanoparticle directly or indirectly through a spacer group; (c) alinker-payload conjugate, wherein the payload is a cytotoxic agent;wherein the linker-payload conjugate is attached to the nanoparticledirectly or indirectly thorough a spacer group; wherein the cytotoxicagent is released upon cleavage of the linker; wherein the linker in thelinker-payload conjugate is selected from a group consisting ofprotease-cleavable linkers, redox-sensitive linkers and pH-sensitivelinkers, wherein the NDC has an average diameter between about 1 nm andabout 10 nm, e.g., between about 3 nm and about 8 nm, or between about 3nm and about 6 nm.

In the NDCs of the present disclosure, the average nanoparticle topayload ratio may range from 1 to 80, such as from 1 to 21 (e.g., 1 to13, or 1 to 12) and the average nanoparticle to targeting ligand ratiomay range from 1 to 50, such as from 1 to 25 (e.g., 1 to 11).

The NDCs of the present disclosure may have an average diameter ofbetween about 1 nm and about 10 nm, e.g., between about 5 nm and about 8nm, between about 3 nm and about 8 nm, or between about 3 nm and about 6nm.

The NDCs of the present disclosure may comprise any suitable dye ordetectable compound. The NDCs of the present disclosure may comprise anysuitable fluorescent compound and/or payload, such as a fluorescentcompound and/or payload disclosed herein. For example, in an NDC of thepresent disclosure, the fluorescent compound may be selected from agroup consisting of Cy5 and Cy5.5, the payload may be selected from agroup consisting of dihydrofolate reductase inhibitors, thymidylatesynthase inhibitors and topoisomerase inhibitors, and the topoisomeraseinhibitor may be selected from a group consisting of SN38, analogs ofSN38, exatecan, and analogs of exatecan. The fluorescent compound may beencapsulated within the nanoparticle (e.g., covalently linked to thesilica core).

The NDCs of the present disclosure can comprise a targeting ligand thatbinds to a folate receptor (FR). The targeting ligand may comprise folicacid, dihydrofolic acid, tetrahydrofolic acid, or any folate receptorbinding derivative of any of the foregoing. It should be understood that“folic acid,” “dihydrofolic acid,” and “tetrahydrofolic acid” mayencompass an amide or an ester of folic acid, dihydrofolic acid, ortetrahydrofolic acid, respectively. For example, “folic acid” may referto the folic acid amide present in the exemplary NDC illustrated in FIG.1 .

The structure of the NDCs of the present disclosure may comprisestructure (S-1′):

wherein Payload is a cytotoxic agent; Linker is selected from a groupconsisting of protease-cleavable linkers, redox-sensitive linkers andpH-sensitive linkers; the silicon atom is a part of the nanoparticle;and X^(1A) and X^(2A) are each independently absent, or a divalentlinker.

The divalent linker X^(1A) may be any suitable divalent linker, forexample, a divalent linker that comprises a chain length of betweenabout 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or acombination thereof), such as between about 5 and about 100 atoms, about5 and about 80 atoms, between about 10 and about 80 atoms, between about10 and about 70 atoms, between about 10 and about 30 atoms, betweenabout 20 and about 30 atoms, between about 30 and about 80 atoms, orbetween about 30 and about 60 atoms.

The divalent linker X^(1A) may be alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene, and can besubstituted or unsubstituted. The divalent linker X^(1A) may comprise acyclic group (e.g., a cyloalkylene, heterocyclylene, arylene, orheteroarylene). For example, the divalent linker X^(1A) may be analkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, orheteroalkynylene group that comprises a cyclic group, such as apiperazine. The divalent linker X^(1A) may comprise a polyethyleneglycol (PEG) moiety, for example, a PEG moiety comprising between about2 and about 20 PEG monomers. The divalent linker X^(1A) may besubstituted with oxo.

The divalent linker X^(1A) may be

The divalent linker X^(2A) may be any suitable divalent linker, forexample, a divalent linker that comprises a chain length of betweenabout 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or acombination thereof), such as between about 5 and about 100 atoms, 5 andabout 80 atoms, between about 10 and about 80 atoms, between about 10and about 70 atoms, between about 10 and about 30 atoms, between about20 and about 30 atoms, or between about 30 and about 60 atoms.

The divalent linker X^(2A) may be alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene, and may besubstituted or unsubstituted. The divalent linker X^(2A) may comprise acyclic group (e.g., a cycloalkylene, heterocyclylene, arylene, orheteroarylene). For example, the divalent linker X^(2A) may be analkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, orheteroalkynylene group that comprises a cyclic group, such as apiperazine. The divalent linker X^(2A) may comprise a polyethyleneglycol (PEG) moiety, for example, a PEG moiety comprising between about2 and about 20 PEG monomers. The divalent linker X^(2A) may besubstituted with oxo.

The divalent linker X^(2A) may be

The NDCs of the present disclosure may comprise structure (S-1″):

wherein P denotes a payload that is a cytotoxic agent; L denotes alinker that is selected from a group consisting of protease-cleavablelinkers, redox-sensitive linkers and pH-sensitive linkers; the siliconatom is a part of the nanoparticle; A^(1A) is —SO₂— or —C(O)—; X^(1B) is—(CH₂)_(a)—NH—C(O)—, —(CH₂)_(a)—NH—SO₂—, —NH—, —(CH₂)_(a),—CH₂—CH₂—(O—CH₂—CH₂)_(a)—NH—C(O)—, —CH₂—CH₂—(O—CH₂—CH₂)_(a)—NH—SO₂—,—(CH₂)_(a)—NH—C(O)—(CH₂)_(a)—(O—CH₂—CH₂)_(a)—NH—C(O)—(CH₂)_(a)—, orabsent; a is an integer of 0-20; y and n are each an integer of 1-20;and Z^(IA) is —O— or —CH₂—.

The NDCs of the present disclosure may comprise structure (S-1

wherein Payload is a cytotoxic agent; Linker is selected from a groupconsisting of protease-cleavable linkers, redox-sensitive linkers andpH-sensitive linkers, and the silicon atom is a part of thenanoparticle.

The NDCs of the present disclosure may comprise structure (S-2′):

wherein Targeting Ligand is selected from the group consisting of folicacid, dihydrofolic acid, tetrahydrofolic acid, and folate receptorbinding derivatives of any of the foregoing; the silicon atom is a partof the nanoparticle, and X^(3A) and X^(4A) are each independentlyabsent, or a divalent linker.

The divalent linker X^(3A) may be any suitable divalent linker, forexample, a divalent linker that comprises a chain length of betweenabout 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or acombination thereof), such as between about 5 and about 100 atoms, about5 and about 80 atoms, 10 and about 80 atoms, between about 10 and about70 atoms, between about 10 and about 30 atoms, between about 20 andabout 30 atoms, between about 30 and about 80 atoms, or between about 30and about 60 atoms.

The divalent linker X^(3A) may be alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene, and may besubstituted or unsubstituted. The divalent linker X^(3A) may comprise acyclic group (e.g., a cycloalkylene, heterocyclylene, arylene, orheteroarylene). For example, the divalent linker X^(3A) may be analkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, orheteroalkynylene group that comprises a cyclic group, such as apiperazine. The divalent linker X^(3A) may comprise a polyethyleneglycol (PEG) moiety, for example, a PEG moiety comprising between about2 and about 20 PEG monomers. The divalent linker X^(3A) may besubstituted with oxo.

The divalent linker X^(3A) may be

The divalent linker X^(4A) may be any suitable divalent linker, forexample, a divalent linker that comprises a chain length of betweenabout 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or acombination thereof), such as between about 5 and about 100 atoms, about5 and about 80 atoms, between about 10 and about 80 atoms, between about10 and about 70 atoms, between about 10 and about 30 atoms, betweenabout 20 and about 30 atoms, between about 30 and about 80 atoms, orbetween about 30 and about 60 atoms.

The divalent linker X^(4A) may be alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene, and may besubstituted or unsubstituted. The divalent linker X^(4A) may comprise acyclic group (e.g., a cycloalkylene, heterocyclylene, arylene, orheteroarylene group). For example, the divalent linker X^(4A) may be analkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, orheteroalkynylene group that comprises a cyclic group, such as apiperazine. The divalent linker X^(4A) may comprise a polyethyleneglycol (PEG) moiety, for example, a PEG moiety comprising between about2 and about 20 PEG monomers. The divalent linker X^(4A) may besubstituted with oxo.

The divalent linker X^(4A) may be

The NDCs of the present disclosure may comprise structure S-2”):

wherein T denotes a targeting ligand selected from the group consistingof folic acid, dihydrofolic acid, tetrahydrofolic acid, and folatereceptor binding derivatives of any of the foregoing; the silicon atomis a part of the nanoparticle; A^(2A) is —SO₂— or —C(O)—; X^(3B) is—(CH₂)_(a)—NH—C(O)—, —(CH₂)_(a)—NH—SO₂—, —NH—, —(CH₂)_(a),—CH₂—CH₂—(O—CH₂—CH₂)_(a)—NH—C(O)—, —CH₂—CH₂—(O—CH₂—CH₂)_(a)—NH—SO₂—,—(CH₂)_(a)—NH—C(O)—(CH₂)_(a)—(O—CH₂—CH₂)_(a)—NH—C(O)—(CH₂)_(a)—, orabsent; a is an integer of 0-20; m and z are each an integer of 2-20;Z^(2A) is —O— or —CH₂—; K is absent,—C(O)—NH—(CH₂)_(j)—NH—CO—CH₂—CH₂—(O—CH₂—CH₂)_(q)—CH₂—CH₂—,—SO₂—NH—(CH₂)_(j)—NH—C(O)—CH₂—CH₂—(O—CH₂—CH₂)q-CH₂—CH₂—,—C(O)—NH—CH₂—CH₂—(O—CH₂—CH₂)q-CH₂—CH₂—,—SO₂—NH—CH₂—CH₂—(O—CH₂—CH₂)_(q)—CH₂—CH₂—,—C(O)-piperazine-C(O)—CH₂—CH₂—(O—CH₂—CH₂)_(q)—CH₂—CH₂—,—SO₂-piperazine-C(O)—CH₂—CH₂—(O—CH₂—CH₂)_(q)—CH₂—CH₂—, j is an integerof 0-10; and q is an integer of 0-10.

The NDCs of the present disclosure may comprise Structure (S-2):

wherein Targeting Ligand is selected from the group consisting of folicacid, dihydrofolic acid, tetrahydrofolic acid, and folate receptorbinding derivatives of any of the foregoing; and the silicon atom is apart of the nanoparticle.

In preferred aspects of structure (S-2), the targeting ligand is FolicAcid.

Structures S-1, S-1′, S-1″, S-2, S-2′, or S-2″ may be present in the NDCat any desired ratio, e.g., a ratio disclosed herein.

The present disclosure also relates to self-immolative linkers andlinker-drug conjugates, and payloads (e.g., drugs), suitable forconjugation to a carrier particle. For example, described herein arelinker-drug conjugates of Formulae (I)—(XII) and (I-B)-(XII-B),including salts thereof, and self-immolative linkers of Formulae(I-A)-(X-A), including salts thereof The carrier particle can be, but isnot limited to, a nanoparticle, a liposome, a nanogel, a nanoring, ananocage, a microsphere, an antibody, an antigen-binding portion of anantibody including single chain antibodies and fragments (e.g., scFv)and/or antibody fragment (e.g., Fab, F(ab′)₂), minibody, or nanobody.The linkers disclosed herein may be useful in the preparation of carrierparticle conjugates, e.g., for drug delivery. Payloads disclosed hereininclude, for example, cytotoxic drugs, including certain analogs of SN38and exatecan. The linkers, linker-drug conjugates, and/or payloadsdisclosed herein may be present in an NDC (e.g., an NDC of the presentdisclosure), or may be present in another suitable carrierparticle-conjugate. The structures of Formulae (I)-(XII), (I-B)-(XII-B),and (IA)-(X-A) are each provided herein. For example, the NDCs of thisdisclosure may comprise linker-payload conjugates of Formulae (I)-(IV),or a salt thereof:

wherein the variables are as described herein.

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a fluorescent compound covalentlyencapsulated within the core of the nanoparticle; a targeting ligand,wherein the targeting ligand is folic acid; a linker-payload conjugate,wherein the linker-payload conjugate is a protease cleavable linker thatis capable of undergoing hydrolysis at the C-terminal end upon proteasebinding thereby releasing the payload from the nanoparticle, wherein theprotease comprises a serine protease or a cysteine protease, wherein thepayload in the linker-payload conjugate is selected from a groupconsisting of SN-38, analog of SN-38, exatecan, and an analog ofexatecan; and wherein the fluorescent compound is Cy5.

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a Cy5 dye covalently encapsulated withinthe core of the nanoparticle; a targeting ligand that binds to folatereceptor, wherein the targeting ligand is folic acid, and wherein thetargeting ligand is attached to the nanoparticle indirectly through aspacer group; a linker-payload conjugate, wherein the linker-payloadconjugate is attached to the nanoparticle indirectly through a spacergroup, wherein the linker-payload conjugate comprises a compoundcomprising the structure

and wherein the NDC has an average diameter between about 1 nm and about10 nm (e.g., between about 1 and about 6 nm).

The NDCs of this disclosure may comprise a linker-payload conjugate ofFormulae (V)-(X), or a salt thereof:

wherein the variables are as described herein.

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a fluorescent compound covalentlyencapsulated within the core of the nanoparticle; a targeting ligand,wherein the targeting ligand is folic acid; a linker-payload conjugate,wherein the linker-payload conjugate is redox-sensitive, wherein thepayload in the linker-payload conjugate is selected from a groupconsisting of SN-38, analog of SN-38, exatecan or an analog of exatecan;and wherein the fluorescent compound is Cy5.

The NDCs of this disclosure may comprise a linker-payload conjugate ofFormulae (XI)-(XII), or a salt thereof.

wherein the variables are as described herein

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a fluorescent compound covalentlyencapsulated within the core of the nanoparticle; a targeting ligand,wherein the targeting ligand is folic acid; a linker-payload conjugate,wherein the linker-payload conjugate is pH-sensitive, wherein thepayload in the linker-payload conjugate is selected from a groupconsisting of SN-38, an analog of SN-38, exatecan, and an analog ofexatecan; and wherein the fluorescent compound is Cy5.

This disclosure also provides a method of treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor expressing tumor),comprising administering to a subject in need thereof an effectiveamount of an NDC described herein. The method may include administrationof NDCs to the subject in need thereof intravenously. In the methods ofthe present disclosure, the subject may have a cancer selected from thegroup consisting of ovarian cancer, endometrial cancer, fallopian tubecancer, cervical cancer, breast cancer (including, e.g., HER2+ breastcancer, HR+ breast cancer, HR− breast cancer, and triple-negative breastcancer), lung cancer (e.g., non-small cell lung cancer (NSCLC),mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML). The NDCs of the presentdisclosure may also be used for targeting tumor associated macrophages,which may be used as a means to modify the immune status of a tumor in asubject. The NDCs of the present disclosure may be used in a method oftreating an advanced, recurrent, or refractory solid tumor.

This disclosure provides use of an NDC for treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor expressing tumor). Theuse may include administration of NDCs intravenously to the subject inneed thereof. In the use of the NDC, the subject may have a cancerselected from the group consisting of ovarian cancer, endometrialcancer, fallopian tube cancer, cervical cancer, breast cancer(including, e.g., HER2+ breast cancer, HR+ breast cancer, HR− breastcancer, and triple-negative breast cancer), lung cancer (e.g., non-smallcell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinalcancer (e.g., esophageal cancer, colon cancer, rectal cancer, andstomach cancer), pancreatic cancer, bladder cancer, kidney cancer, livercancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer,prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g.,pediatric AML), and chronic myelogenous leukemia (CML). In the use ofthe NDC, the cancer may be an advanced, recurrent, or refractory solidtumor.

This disclosure provides NDCs for use in the manufacture of a medicamentfor treating folate receptor (FR)-expressing cancer (e.g., a folatereceptor expressing tumor). The use in the manufacture of a medicamentmay include administration of NDCs intravenously to the subject in needthereof The use in the manufacture of a medicament may includeadministration of NDCs to a subject, wherein the subject has a cancerselected from the group consisting of ovarian cancer, endometrialcancer, fallopian tube cancer, cervical cancer, breast cancer(including, e.g., HER2+ breast cancer, HR+ breast cancer, HR− breastcancer, and triple-negative breast cancer), lung cancer (e.g., non-smallcell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinalcancer (e.g., esophageal cancer, colon cancer, rectal cancer, andstomach cancer), pancreatic cancer, bladder cancer, kidney cancer, livercancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer,prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g.,pediatric AML), and chronic myelogenous leukemia (CML). The NDCs of thepresent disclosure may be used in the manufacture of a medicament fortreating an advanced, recurrent, or refractory solid tumors.

This disclosure also relates to a pharmaceutical composition comprisingan NDC and a pharmaceutically acceptable excipient. The pharmaceuticalcompositions disclosed herein may be used for treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor expressing tumor).

In some embodiments, an NDC of the present disclosure does not comprisea structure of Formula (NP-2):

wherein x and y are each independently an integer of 0 to 20 (e.g., 4and 3, respectively).

In some embodiments, an NDC of the present disclosure does not comprisea structure of Formula (NP-3):

wherein x and y are each independently an integer of 0 to 20 (e.g., 4and 9, respectively).

In some embodiments, an NDC comprising a structure of Formula (NP-2)does not comprise a structure of Formula (NP-3). In some embodiments, anNDC comprising a structure of Formula (NP-3) does not comprise astructure of Formula (NP-2).

In some embodiments, an NDC of the present disclosure does not comprisea structure of Formula (S-1a):

wherein the silicon atom is a part of the nanoparticle. In someembodiments, an NDC of the present disclosure does not comprise astructure of Formula (S-2a):

wherein the silicon atom is a part of the nanoparticle.

In some embodiments, an NDC comprising a structure of Formula (S-1a)does not comprise a structure of Formula (S-2a). In some embodiments, anNDC comprising a structure of Formula (S-2a) does not comprise astructure of Formula (S-1a).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical structure of a representativenanoparticle-drug conjugate (NDC).

FIG. 2 depicts a flow chart for the synthesis of an exemplaryfunctionalized nanoparticle (dibenzocyclooctyne (DBCO)-functionalizedC′Dot).

FIG. 3 depicts a flow chart for the synthesis of an exemplary NDC(FA-CDC) comprising a C′Dot functionalized with folic acid (FA) andexatecan.

FIG. 4 illustrates a representative UV-Vis absorbance spectrum of anexemplary functionalized nanoparticle (DBCO-functionalized C′Dot). Theabsorption peak at 648 nm correspond to the Cy5 dye that is covalentlyencapsulated within the core of the nanoparticle. The absorption peaksaround 270 to 320 nm correspond to DBCO groups on the nanoparticle.

FIG. 5 illustrates a representative UV-Vis absorbance spectrum of anexemplary NDC (folic acid (FA)-functionalized C′Dot (FA-CDC)). Theabsorption peak at 648 nm correspond to the Cy5 dye that is covalentlyencapsulated within the core of the nanoparticle. The absorption peaksaround 330 to 400 nm correspond to exatecan on the nanoparticle.

FIG. 6 depicts a fluorescence correlation spectroscopy (FCS) correlationcurve of an exemplary NDC (folic acid (FA)-functionalized drug-linkerconjugated C′Dot (FA-CDC)) that is fitted by a single-modal FCScorrelation function. Average hydrodynamic diameter was obtained viafitting the FCS correlation curve.

FIG. 7 depicts a chromatogram showing the elution of an exemplary NDC(folic acid (FA)-functionalized drug-linker conjugated C′Dot (FA-CDC))by a gel permeation chromatography (GPC). The elution of FA-CDC (stripedline under the curve) is compared to the elution time of proteinstandards with varying molecular weight (dashed line).

FIG. 8 depicts a reversed phase HPLC chromatogram (RP-HPLC) of apurified exemplary NDC (folic acid (FA)-functionalized drug-linkerconjugated C′Dot (FA-CDC)) at 330 nm. This wavelength can be used tomonitor both the NDC and impurities that may be present after thesynthesis or due to any degradation of the NDC.

FIGS. 9A-9B illustrate the UV-Vis absorbance spectra of exemplarylinker-payload conjugates. FIG. 9A depicts the absorbance spectrum of arepresentative linker-payload conjugate with SN38 as payload. FIG. 9Bdepicts the absorbance spectrum of a representative linker-payloadconjugate with exatecan as payload. Both SN38 and exatecan payloads haveabsorption maxima around 360 nm.

FIGS. 10A-10B illustrate a representative HPLC chromatographs of anexemplary NDC loaded with folic acid as targeting ligand andlinker-payload conjugates prepared using linker-payload conjugateprecursor Compound 202, from Example 33). FIG. 10A depicts arepresentative HPLC chromatograph at 360 nm of a non-cleaved NDC showinga single peak at elution time around 6.3 min which corresponds to thenon-released payload retained on the NDC. FIG. 10B depicts arepresentative HPLC chromatograph at 360 nm of a cleaved NDC, showing anadditional peak at elution time around 3 to 4 min which corresponds tothe released payload. The area under curve (AUC) of the released payloadand the retained payload were used to calculate the percentage ofreleased payload.

FIGS. 11A-11C are plots illustrating a representative drug releasinganalysis of exemplary NDCs loaded with folic acid as targeting ligandand protease (cathepsin-B) cleavable linker-payload conjugates, atdifferent time points after incubation with cathepsin-B enzyme. FIG. 11Adepicts the RP-HPLC chromatograph of an exemplary NDC prepared usingCompound 89 from Example 10, at different time points after incubationwith cathepsin-B enzyme. FIG. 11B depicts the RP-HPLC chromatograph ofan exemplary NDC prepared using Compound 158 from Example 25, atdifferent time points after incubation with cathepsin-B enzyme. FIG. 11Cdepicts the RP-HPLC chromatograph of an exemplary NDC prepared usingCompound 202 from Example 33, at different time points after incubationwith cathepsin-B enzyme.

FIGS. 12A-12C are plots illustrating a drug releasing kinetics analysisof exemplary NDCs loaded with protease (cathepsin-B) cleavablelinker-payload conjugates at different time points after incubation withcathepsin-B, and depicts the time for half of the payloads to bereleased, i.e. T_(1/2). FIG. 12A depicts the T_(1/2) as 2.9 hours for anexemplary NDC prepared using Compound 89 from Example 10. FIG. 12Bdepicts the T_(1/2) as 2.6 hours for an exemplary NDC prepared usingCompound 158 from Example 25. FIG. 12C depicts the T_(1/2) as 1.4 hoursfor an exemplary NDC prepared using Compound 202 from Example 33.

FIG. 13A depicts the competitive binding of an exemplary NDC (folic acid(FA)-functionalized drug-linker conjugated C′Dot (FA-CDC)) in a FR alphapositive (KB) cell line, when compared with free folic acid.

FIG. 13B depicts the flow cytometry median fluorescence intensity (MFI)of an exemplary NDC (folic acid (FA)-functionalized drug-linkerconjugated C′Dot (FA-CDC)) in different cell lines, including a FR alphapositive cell line (KB), a FR alpha negative cell line (TOV), and a FRreceptor blocked cell line (KB) (100 nM, 4° C., 60 min). The activetargeting of FA-CDC can be fully blocked by incubating with the presenceof 1 mM free folic acid. The NDC used in the study was prepared withcompound 170 that is described in Example 27; each NDC contained 15folic acid moieties, and 40 linker-drug conjugate moieties.

FIG. 14 depicts the flow cytometry of representative NDCs (two folicacid (FA)-functionalized drug-linker conjugated C′Dot (FA-CDCs)) in KBcell line with varied folic acid ligand density (either an average of 0,12, or 25 folic acid molecules per nanoparticle). The linker-drugconjugate precursor used to prepare the representative NDCs is Compound202 described in Example 33. Blocking in the blocking group was achievedusing 1 mM of free folic acid. A CDC with no folic acid, but same amountof drug linker conjugates, was used as the negative control group.

FIG. 15 depicts the flow cytometry of representative NDCs (three folicacid (FA)-functionalized drug-linker conjugated C′Dots (FA-CDCs) in KBcell line with varied drug per particle ratio (DPR). The linker-drugconjugate precursor used to prepare the representative NDCs used in thestudy is described in Example 33 (Compound 202). Blocking in theblocking group was achieved with 1 mM of free Folic Acid. Allrepresentative NDCs comprised between 12 and 22 folic acid moieties. TheNDCs with high drug-particle ratios (DPRs) comprise between 35 and 50linker-drug conjugate groups. FA-CDCs with medium DPRs comprise between17 and 25 linker-drug conjugate groups. FA-CDCs with low DPRs havebetween 5 and 10 linker-drug conjugate groups. A CDC with no folic acid,and 17 to 25 drug linkers, was used as the negative control group.

FIG. 16 depicts the flow cytometry of a representative NDC (folic acid(FA)-functionalized drug-linker conjugated C′Dot (FA-CDC)) at 1 nM thatwas pre-incubated with varied amounts of human plasma for 24 hours.Blocking in the blocking group was achieved with 1 mM of free folicacid. The linker-drug conjugate precursor used to prepare the NDC usedin the study is described in Example 33 (Compound 202), the number offolic acid ligands on the NDC is 15; the number of linker-drugconjugates on the NDC is 25). An NDC with no folic acid, but same amountof drug linkers was used as the negative control group.

FIG. 17 shows the confocal microscopy images of an exemplary NDC (folicacid (FA)-functionalized drug-linker conjugated C′Dot (FA-CDC)) inKB(++++) and TOV−112D(−) cell lines at 1 hour and 24 hours. Blocking inthe blocking group was achieved with 0.1 mM of free folic acid. Thelinker-drug conjugate precursor used to prepare the NDC used in thestudy is Compound 87 described in Example 10, the number of folic acidligands on the NDC is 12; and the number of linker-drug conjugates onthe NDC is 40). The lysosome was stained by using LysoTracker® Green,which is a green-fluorescent dye for labeling and tracking acidicorganelles in live cells. With color images (not shown), the CDC appearsred, the lysosome appears green, and the nucleus appears blue, due tofluorescence.

FIG. 18 is an image comparing the Z-stack confocal microscopic imagingof KB tumor spheroids treated with an exemplary folate-receptor(FR)-targeting NDC, a payload-free FR-targeting nanoparticle (FA-C′Dot),a FR-targeting ADC, or the corresponding payload-free FR-targetingantibody, at 37° C. for 4 hours. The FR-targeting NDC was prepared usingthe drug-linker conjugate precursor Compound 220 (Example 33). Scalebar: 200 m.

FIG. 19A depicts a representative maximum intensity projection (MIP)PET/CT imaging of healthy nude mice injected with ⁸⁹Zr-DFO-FA-CDC at 1,24, 48 and 72 hours post-injection.

FIG. 19B illustrates the biodistribution pattern of ⁸⁹Zr-DFO-FA-CDC inhealthy nude mice at 2 and 24 hour post-injection (n=3). The linker-drugconjugate precursor used to prepare the NDC used in the study isdescribed in Example 33 (Compound 202), the number of folic acid ligandson each NDC is 12; and the number of linker-drug conjugates on each NDCis 25).

FIGS. 20A-20F depicts the in vivo tumor growth inhibition studies of sixexemplary folate receptor targeting NDCs (NDCs A-F) in KB tumor-bearingmice (n=7). NDC A comprises about 19 drug-linker conjugate groups,prepared using the drug-linker conjugate precursor Compound 342 (Example57), and about 18 folic acid ligands per nanoparticle. NDC B comprisesabout 25 drug-linker groups, prepared using the drug-linker conjugateprecursor Compound 87 (Example 10), and about 15 folic acid ligands pernanoparticle. NDC C comprises about 19 drug-linker conjugate groups,prepared using the drug-linker conjugate precursor Compound 158 (Example25), and about 13 folic acid ligands per nanoparticle. NDC D comprisesabout 25 drug-linker conjugate groups, prepared using the drug-linkerconjugate precursor Compound 202 (Example 33), and about 12 folic acidligands per nanoparticle. NDC E comprises about 17 drug-linker conjugategroups, prepared using the drug-linker conjugate precursor Compound 418(Example 70), and about 17 folic acid ligands per nanoparticle. NDC Fcomprises about 23 drug-linker conjugate groups, prepared using thedrug-linker conjugate precursor Compound 430 (Example 74), and about 20folic acid ligands per nanoparticle.

FIGS. 21A-21B depict the IC₅₀ curves of an exemplary NDC inirinotecan-resistant and naïve KB cells, compared to non-conjugatedirinotecan. FIG. 21A shows the IC₅₀ curves of irinotecan in regular KBcells (naïve cells), and in KB cells treated four times with irinotecan(irinotecan-resistant cells). FIG. 21B shows the IC₅₀ curves of theexemplary NDC in the naïve cells, and in the irinotecan-resistant cells.The linker-drug conjugate precursor used to prepare the exemplary NDC ofthis study is described in Example 33 (Compound 202).

FIGS. 22A-22B depict the IC₅₀ curves of an exemplary NDC inexatecan-resistant and naïve KB cells, compared to non-conjugatedexatecan. FIG. 21A shows the IC₅₀ curves of exatecan in regular KB cells(naïve cells), and in KB cells treated four times or seven times withexatecan (exatecan-resistant cells). FIG. 22A shows the IC₅₀ curves ofthe exemplary NDC in the naïve cells and in the exatecan-resistant cells(4-cycle and 7-cycle pretreatment). The linker-drug conjugate precursorused to prepare the exemplary NDC of this study is described in Example33 (Compound 202).

FIG. 23 provides a table demonstrating the cytotoxicity of exemplaryfolate receptor targeting NDCs (“FA-CDC”) with varying drug-to-particleratios (DPRs), in different FR-alpha overexpressing cancer cell lines,compared to non-conjugated exatecan. The linker-drug conjugate precursorused to prepare the exemplary NDCs of this study is described in Example33 (Compound 202).

FIG. 24 provides a table showing the cytotoxicity of an exemplary NDC invarious 3D patient-derived platinum-resistant tumor spheroids. Thelinker-drug conjugate precursor used to prepare the exemplary NDC ofthis study is described in Example 33 (Compound 202).

FIGS. 25A-25D provide flow cytometry histograms demonstrating thespecific folate receptor (FR) alpha targeting capability of an exemplaryFR-targeting NDC (prepared using the drug-linker conjugate precursorCompound 202 from Example 33) to both the IGROV-1 (FR alpha positivehuman ovarian cancer) and the engineered AML MV4;11 cell lines thatoverexpress FR alpha. FIG. 25A is the flow cytometry histogram of the FRtargeting NDC (10 nM) and non-targeting NDC (negative control; 10 nM) inIGROV-1 cell line. FIG. 25B is the flow cytometry histogram of anti-FRalpha antibody-PE and isotype antibody-PE (negative control) in IGROV-1cell line. FIG. 25C is the flow cytometry histogram of the FR targetingNDC (10 nM) and non-targeting NDC (negative control; 10 nM) inengineered AML MV4;11 cell lines that overexpress FR alpha. FIG. 25D isthe flow cytometry histogram of anti-FR alpha antibody-PE and isotypeantibody-PE (negative control) in engineered AML MV4;11 cell lines thatoverexpress FR alpha.

FIGS. 26A-26B are graphs illustrating the in vitro cytotoxic activity ofan exemplary NDC (prepared using the drug-linker conjugate precursorCompound 202 described in Example 33) in IGROV-1 (FR alpha positivehuman ovarian cancer) cell line (FIG. 26A) and engineered AML MV4;11cell lines that overexpress FR alpha (FIG. 26B) using non-targeted NDCas negative control.

FIG. 27 is a graph providing the bodyweight change of FR alphaoverexpressing AML mice over time after treatment with normal saline oran exemplary NDC (prepared using the drug-linker conjugate precursorCompound 202 from Example 33) at three different dose regimens (0.33mg/kg, Q3D×6 (denoted with squares); 0.50 mg/kg, Q3D×3 (denoted withdiamonds); or 0.65 mg/kg, Q3D×3 (denoted with triangles)).

FIG. 28 provides images from in vivo bioluminescence imaging (BLI) of FRalpha overexpressing AML mice treated with normal saline or an exemplaryNDC (prepared using the drug-linker conjugate precursor Compound 202from Example 33) at three different dose regimens (0.33 mg/kg, Q3D×6);0.50 mg/kg, Q3D×3; or 0.65 mg/kg, Q3D×3).

FIG. 29 is a graph providing the quantitative in vivo bioluminescenceimaging analysis of FR alpha overexpressing AML mice treated with normalsaline or an exemplary NDC (prepared using the drug-linker conjugateprecursor Compound 202 from Example 33) at three different dose regimens(0.33 mg/kg, Q3D×6); 0.50 mg/kg, Q3D×3; or 0.65 mg/kg, Q3D×3).

FIG. 30 is a graph indicating the leukemia detected in bone marrowaspiration at Day 42 post-leukemia cell injection, obtained from micetreated with normal saline or an exemplary NDC (prepared using thedrug-linker conjugate precursor Compound 202 from Example 33) at threedifferent dose regimens (0.33 mg/kg, Q3D×6); 0.50 mg/kg, Q3D×3; or 0.65mg/kg, Q3D×3).

FIG. 31 is an illustration of the timeline used for preparing FR alphaoverexpressing AML mice, and dosing the mice with an exemplary NDC(prepared using the drug-linker conjugate precursor Compound 202 fromExample 33) at three different dose regimens (0.33 mg/kg, Q3D×6); 0.50mg/kg, Q3D×3; or 0.65 mg/kg, Q3D×3), and imaging the mice withbioluminescent imaging (BLI). Each day of dosing is denoted by atriangle (i.e., on days 46, 49, and 52 for all dose groups, and also ondays 55, 58, and 62 for the 0.33 mg/kg Q3D×6 dose group).

FIGS. 32A-32B are graphs demonstrating the stability of exemplary NDCsdisclosed herein. FIG. 32A compares the stability of an NDC producedusing a diene-based functionalized nanoparticle (i.e., based on theprotocol outlined in the Examples herein), and a comparative NDCproduced using an amine-based functionalized nanoparticle, in humanserum at 37° C., over 7 days. FIG. 32B compares the stability of the NDCproduced using a diene-based bifunctional precursor, and the comparativeNDC produced using an amine-based bifunctional precursor, in mouse serumat 37° C., over 7 days.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are carrier particle drug conjugates, which comprise acarrier particle (e.g., nanoparticle) that is conjugated to a drug(sometimes referred to herein as a payload) and optionally a targetingligand, and/or other compound. The carrier particle-drug conjugatesdisclosed herein can be used for delivering a drug to a biologicaltarget (e.g., for targeted delivery to a cancer cell or tumor). The drugmay be linked to the carrier particle via a self-immolative linker,which can be selectively cleaved, e.g., within a cancer cell or tumor.

Carrier particle drug conjugates disclosed herein includenanoparticle-drug conjugates (NDC), which comprise a nanoparticle (e.g.,a silica nanoparticle, such as a multi-modal silica-based nanoparticle)that allows conjugation to targeting ligands and to cytotoxic payloads,for the detection, prevention, monitoring and/or treatment of a disease,such as cancer.

This disclosure provides compositions and methods of using ananoparticle-drug conjugate (NDC) comprising: a nanoparticle; atargeting ligand that binds to a folate receptor, and a linker-payloadconjugate.

The conjugation of both targeting ligands and linker-drug conjugates tothe carrier particle (e.g., nanoparticle) can be achieved via a highlyefficient “click chemistry” reaction, which is fast, simple to perform,versatile, and results in high product yields. The payload may be acytotoxic agent that is attached to the carrier particle (e.g.,nanoparticle) via a cleavable linker group, such as a payload describedherein. The cleavable linker group can be cleaved when the carrierparticle drug conjugate (e.g., NDC) is internalized in a cancer cell(e.g., in a tumor cell), and can be cleaved in the endosome or lysosomalcompartment of a cancer cell (e.g., a tumor cell), causing release ofthe active cytotoxic agent from the carrier particle-drug conjugate(e.g., NDC).

The NDCs disclosed herein provide an optimal platform for drug deliverydue to their physical properties. For example, the NDCs comprisenanoparticles that are ultrasmall in diameter (e.g. with averagediameter between about 1 nm and about 10 nm, such as between about 5 nmand about 8 nm) and benefit from enhanced permeability and retention(EPR) effects in tumor microenvironments, while retaining desiredclearance and pharmacokinetic properties.

The carrier particle-drug conjugates (e.g., NDCs) described herein havecertain advantages over other drug delivery platforms (e.g., ADCs suchas FR-targeted ADCs, and FR-targeted small molecule drugs (e.g.,chemotherapeutics)). For example, a single NDC of the present disclosuremay include up to about 80 drug molecules on each nanoparticle. Incontrast, in conventional ADCs, only about 4 to 8 therapeutic/drugmolecules can be attached to the antibody, and conventional FR-targetedsmall molecule drug conjugates are limited to only a singletherapeutic/drug molecule. Thus, the NDCs described herein can carry atleast 10 times more drug molecules (either a single type of drug, ormultiple types of drugs, such as drugs having different mechanisms ofaction) on a single nanoparticle, relative to conventional drug deliveryplatforms, and deliver a relatively higher drug load to cells.

While conventional FR-targeted drug-delivery platforms, such as ADCs andFR-targeted small molecular chemotherapeutics, usually exhibit highpotency in cancer cells with high receptor expression level, theirefficacy in cancer cells with medium or low FR expression level islimited. In contrast, the carrier particle drug conjugates (e.g., NDCs)of the present disclosure can effectively target cancer cells with bothhigh and low FR expression levels, and provide potent therapy forcancers that have low FR expression (see, e.g., FIG. 23 and associatedassay described in the Examples).

Without wishing to be bound by any particular theory of mechanism, it isbelieved that, because the NDCs disclosed herein can include multipleFR-targeting ligands on a single nanoparticle, there is a multivalent oravidity effect on binding to several FRs on the cell surface. Incontrast, a single ADC generally can only bind to up to two FRs on thecell surface, and a single FR-targeted chemotherapy drug can only bindto one FR on the cell surface. Thus, the multivalent effect of theFR-targeted NDCs of the present disclosure can significantly enhance thebinding of NDC to cells that express FR, leading to improved targetingefficiency and therapeutic outcomes. This multivalent effect can alsorender the NDCs of the present disclosure suitable for treating cancersthat have low FR-expression, that cannot be effectively treated usingconventional FR-targeted drug delivery platforms, such as ADCs orFR-targeted chemotherapy drugs.

The efficacy of ADCs in solid tumor treatment is usually greatly limitedby their poor tumor penetration. In contrast, the FR-targeted NDCsdisclosed herein exhibit highly effective tumor penetration, permittingthe delivery of therapeutics throughout a tumor followingadministration, which improves therapeutic outcomes in treating solidtumors, relative to the use of ADCs.

The NDCs of the present disclosure have a smaller size than conventionaldrug delivery platforms, such as ADCs. Notably, the NDCs of the presentdisclosure are smaller than the particle size cut off for renalclearance, permitting the NDC to be renally clearable. As a result, NDCsthat are administered to a subject but do not enter a cancer cell (i.e.,non-targeted NDCs) can be rapidly cleared from the body via renalelimination. This target-and/or-clear approach reduces the toxicity ofNDCs as compared to conventional drug delivery platforms, such as ADCs,and prevents undesirable accumulation of the NDCs (or their payloads) inhealthy tissues or organs.

The NDCs of the present disclosure exhibit improved biodistribution thanconventional drug delivery platforms, such as ADCs, resulting in reducedside effect and toxicity.

Carrier Particles

Disclosed herein are carrier particles suitable for conjugation to adrug and/or targeting ligand, to provide carrier particledrug-conjugates. The carrier particle can be, but is not limited to, ananoparticle, a liposome, a nanogel, a nanoring, a nanocage, amicrosphere, an antibody, an antigen-binding portion of an antibody.

In some aspects, this disclosure relates to NDCs comprising ananoparticle. The nanoparticle may be a silica nanoparticle. Thenanoparticle may comprise a silica-based core and a silica shellsurrounding at least a portion of the core.

Alternatively, the nanoparticle may have only the core and no shell. Thecore of the nanoparticle may contain the reaction product of a reactivefluorescent compound and a co-reactive organo-silane compound. Forexample, the core of the nanoparticle may contain the reaction productof a reactive fluorescent compound and a co-reactive organo-silanecompound, and silica. In preferred aspects of the present disclosure,the nanoparticle is a core-shell particle.

The diameter of the nanoparticle core may be from about 0.5 nm to about100 nm, from about 0.1 nm to about 50 nm, from about 0.5 nm to about 25nm, from about 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm.For example, the diameter of the core may be from about 3 nm to about 8nm, or 3 nm to about 6 nm, e.g., the diameter of the core may be fromabout 3 nm to about 4 nm, about 4 nm to about 5 nm, about 5 nm to about6 nm, about 6 nm to about 7 nm, or about 7 nm to about 8 nm.

The shell of the nanoparticle can be the reaction product of a silicaforming compound, such as a tetraalkyl orthosilicate, for exampletetraethyl orthosilicate (TEOS). The shell of the nanoparticle may havea range of layers. For example, the silica shell may be from about 1 toabout 20 layers, from about 1 to about 15 layers, from about 1 to about10 layers, or from about 1 to about 5 layers. For example, the silicashell may comprise from about ito about 3 layers. The thickness of theshell may range from about 0.5 nm to about 90 nm, from about 0.5 nm toabout 40 nm, from about 0.5 nm to about 20 nm, from about 0.5 nm toabout 10 nm, or from about 0.5 nm to about 5 nm, e.g., about 1 nm, about2 nm, about 3 nm, about 4 nm, or about 5 nm. For example, the thicknessof the silica shell may be from about 0.5 nm to about 2 nm. The silicashell of the nanoparticle may cover only a portion of nanoparticle orthe entire particle. For example, the silica shell may cover about 1 toabout 100 percent, from about 10 to about 80 percent, from about 20 toabout 60 percent, or from about 30 to about 50 percent of thenanoparticle. For example, the silica shell may cover about 50 to about100 percent.

The silica shell can be either solid, i.e., substantially non-porous,meso-porous, semi-porous, or the silica shell may be porous. The silicananoparticle can be either solid, i.e., substantially non-porous,meso-porous, semi-porous, or the silica nanoparticle may be porous. Incertain aspects of the present disclosure, the silica shell is porous.In some aspects, the nanoparticle is a non-mesoporous nanoparticle,e.g., a non-mesoporous silica nanoparticle, such as a non-mesoporoussilica core-shell nanoparticle.

The surface of the carrier particle, e.g., nanoparticle, may be modifiedto incorporate at least one functional group. An organic polymer may beattached to the carrier particle, e.g., nanoparticle, and can bemodified to incorporate at least one functional group by any knowntechniques in the art. The functional groups can include, but are notlimited to, dibenzocyclooctyne (DBCO), maleimide, N-hydroxysuccinimide(NHS) ester, a diene (e.g., cyclopentadiene), an amine, or a thiol. Forexample, a bifunctional group comprising a silane at one terminus, and aDBCO, maleimide, NHS ester, diene (e.g., cyclopentadiene), amine, orthiol at the other terminus, may be condensed onto the surface of asilica nanoparticle via the silane group. The incorporation of thefunctional group can also be accomplished through known techniques inthe art, such as using “click chemistry,” amide coupling reactions,1,2-additions such as a Michael addition, or Diels-Alder (2+4)cycloaddition reactions. This incorporation allows attachment of varioustargeting ligands, contrast agents and/or therapeutic agents to thecarrier particle, e.g., nanoparticle.

The organic polymers that may be attached to the carrier particle, e.g.,nanoparticle include, but are not limited to, poly(ethylene glycol)(PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid(PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA),polyvinyl acetate (PVA), and combinations thereof. In preferred aspectsof the present disclosure, the organic polymer is poly(ethylene glycol)(PEG).

In preferred aspects of the present disclosure, the surface of thenanoparticle is functionalized. For example, the surface of thenanoparticle can have functional groups other than those resulting fromthe synthesis of the nanoparticles (e.g., —OH groups (resulting fromterminal Si—OH groups on a nanoparticle surface) and PEG groups(resulting from Si-PEG groups on the nanoparticle surface). Suchfunctionalization and functionalization methods are known in the art.

The nanoparticle may comprise a non-pore surface and a pore surface. Inan embodiment, at least a portion of the individual nanoparticlenon-pore surface and at least a portion of the individual nanoparticlepore surface are functionalized. In an embodiment, at least a portion ofthe nanoparticle non-pore surface and the at least a portion of the poresurface have different functionalization. The pore surface is alsoreferred to herein as the interior surface. The nanoparticles may alsohave a non-pore surface (or non-porous surface). The non-pore surface isalso referred to herein as the exterior nanoparticle surface.

The pore surface (e.g., at least a portion of the pore surface) and/orthe non-pore surface (e.g., at least a portion of the non-pore surface)of the nanoparticle can be functionalized. For example, thenanoparticles can be reacted with compounds such that a functional groupof the compound is presented on (e.g., covalently bonded to) the surfaceof the nanoparticle. The surface can be functionalized with hydrophilicgroups (e.g., polar groups such as ketone groups, carboxylic acid,carboxylate groups, and ester groups), which provide a surface havinghydrophilic character, or hydrophobic groups (e.g., nonpolar groups suchas alkyl, aryl, and alkylaryl groups), which provide a surface havinghydrophobic character. Such functionalization is known in the art. Forexample, diethoxydimethylsilane (DEDMS) can be condensed on at least aportion of the pore surface such that the pore surface has hydrophobiccharacter, allowing increased loading performance of a hydrophobiccytotoxic payload relative to nanoparticles that are not functionalizedso.

In preferred aspects of the present disclosure, the surface of thenanoparticle is at least partially functionalized with polyethyleneglycol (PEG) groups. The attachment of PEG to the nanoparticle may beaccomplished by a covalent bond or a non-covalent bond, such as by ionicbond, hydrogen bond, hydrophobic bond, coordination, adhesive, andphysical absorption.

In certain aspects, the PEG groups are attached (e.g., covalentlyattached) to the surface of the nanoparticle. In a core-shellnanoparticle, the PEG groups are covalently bonded to the silica at thesurface of the shell via a Si—O—C bond and or to the silica in the core.In a core nanoparticle, the PEG groups are covalently bonded to thesilica in the core.

In preferred aspects, the nanoparticle is a core-shell nanoparticle,wherein the PEG groups are covalently bonded to the silica at thesurface of the shell via a Si—O—C bond. The PEG groups on thenanoparticle surface can prevent adsorption of serum proteins to thenanoparticle in a physiological environment (e.g., in a subject), andmay facilitate efficient urinary excretion and decrease aggregation ofthe nanoparticle (see, e.g., Bums et al. “Fluorescent silicananoparticles with efficient urinary excretion for nanomedicine”, NanoLetters, 2009, 9 (1), 442-448).

The PEG groups may be derived from PEG polymer having a molecular weight(Mw) of 400 g/mol to 2000 g/mol, including all integer g/mol values andranges therebetween. In an embodiment, the PEG groups are derived fromPEG polymer having a Mw of 460 g/mol to 590 g/mol, which contain 6 to 9ethylene glycol units. In various embodiments, the nanoparticles are atleast 50%, at least 75%, at least 90%, or at least 95% functionalizedwith PEG groups. In an embodiment, the nanoparticles are functionalizedwith PEG groups with the maximum number of PEG groups such that, thepores remain accessible (e.g., the pores can be functionalized). In anembodiment, the pore surface is a silica surface having terminal silanol(Si—OH) groups.

A polyethylene glycol unit disclosed herein may be functionalized with afunctional group, for example, a “click chemistry” group such asdibenzocyclooctyne (DBCO) or azide, a diene (e.g., cyclopentadiene), amaleimide, an NHS ester, an amine, a thiol, or an activated acetylenemoiety such as

While DBCO can be used, the functional group may also be another alkyne,such as another strained alkyne (e.g., DIBO or a derivative thereof, ora derivative of DBCO). Also, the functional group may be a nitrone or anitrile oxide.

Alternatively, or in addition to the foregoing, a functional group canbe introduced to a carrier particle, e.g., nanoparticle, withoutnecessarily requiring a PEG group. For example, a nanoparticle may befunctionalized with a functional group such as a “click chemistry”group, e.g., dibenzocyclooctyne (DBCO) or azide, a diene (e.g.,cyclopentadiene), a maleimide, an NHS ester, an amine, a thiol, or anactivated acetylene moiety such as

that may comprise any suitable linker, or may have no linker.

For example, a DBCO-functionalized linker may be introduced to ananoparticle (e.g., a PEGylated C′Dot) by reacting the silane group on aDBCO-linker-silane compound with a silanol group on the surface of thenanoparticle (e.g., under the PEG layer on the C′Dot surface).Similarly, a diene-functionalized precursor (e.g.,cyclopentadiene-functionalized precursor) may be introduced to ananoparticle (e.g., a PEGylated C′Dot) by reacting the silane group on adiene-silane precursor compound with a silanol group on the surface ofthe nanoparticle (e.g., under the PEG layer on the C′Dot surface),providing a nanoparticle functionalized with a diene; followed byfunctionalizing the diene on the nanoparticle with a second precursorthat comprises a group reactive with the diene (e.g., a dienophile), andanother reactive group (e.g., DBCO), thereby providing a nanoparticlefunctionalized with the another reactive group (e.g., DBCO) via adienophile. The linker group in the DBCO-linker-silane or diene-silanecan comprise any structure (or sub-structure), including but not limitedto PEG, a carbon chain, (e.g., alkylene), a heteroalkylene group, or thelike. A diene-functionalized linker covalently attached to a carrierparticle, e.g., nanoparticle, may be further modified, e.g., by reactionwith a DBCO-functionalized group. For example, the diene-functionalizedlinker covalently attached to a nanoparticle may be contacted with aDBCO-linker-maleimide compound (or other suitableDBCO-linker-dienophile), to form a cycloadduct between the diene andmaleimide, resulting in a functionalized nanoparticle comprising DBCOgroups attached to its surface, e.g., using cycloaddition chemistry,such as a Diels-Alder cycloaddition.

Functionalization (e.g., with one of the aforementioned functionalgroups, such as DBCO or cyclopentadiene) facilitates the conjugation ofsuitably functionalized FR-targeting ligands (such asazide-functionalized FR-targeting ligands) and/or functionalized drugpayloads (such as azide-functionalized drug payloads) to the carrierparticle, e.g., nanoparticle, by a coupling reaction, e.g., via clickchemistry, (3+2) cycloaddition reactions, amide coupling, or Diels-Alderreaction. This functionalization approach also improves the versatilityof the formulation chemistry and the stability of the FR-targeted NDCconstructs.

An advantage of the carrier particle-drug conjugates (e.g., NDCs)disclosed herein is that they can be prepared using relatively stablelinker or spacer groups, or precursors thereof The linker or spacergroups, or their precursors, can avoid premature or undesired cleavage,which can occur using other linkers or precursors. For example, certainmethods of functionalizing carrier particles (e.g., nanoparticles)employ amine-silane precursors (to provide amine-functionalized carrierparticles), that are subsequently modified at the amine groups toconjugate other moieties to the carrier particle. However, theamine-silane precursors can be unstable and can self-condense duringreaction, causing undesired aggregation. The aggregates can be verydifficult to separate from the functionalized carrier particles.Additionally, the amine groups on the surface of the carrier particlecan promote undesired reactivity, that may lead to premature release ofthe payload, or undesired release of the targeting ligand, and lead toinstability of the functionalized carrier particles (e.g.,nanoparticles).

The carrier particle drug conjugates (e.g., NDCs) disclosed herein canbe produced using relatively stable precursors, and the resultingfunctionalized carrier particles (e.g., nanoparticles, e.g., NDCs) arestable and highly pure. For example, the functionalized nanoparticles orNDCs disclosed herein can be prepared with a silane-diene precursor(such as a silane-cyclopentadiene precursor), to afford a nanoparticlefunctionalized with one or more diene groups. The diene groups may thenbe reacted with a second precursor, such as a dienophile-containingprecursor (e.g., a PEG-maleimide derivative, e.g., aDBCO-PEG-maleimide), causing a stable cycloadduct to form. The resultingfunctionalized nanoparticle, comprising the cycloadduct, may optionallybe reacted with one or more subsequent precursors (such as targetingligand precursors and/or payload-linker conjugate precursors describedherein), to further functionalize the nanoparticle. The diene-silaneprecursors, and the cycloadducts that are produced, do not exhibit theundesired qualities of other functionalized nanoparticles, e.g., theyhave relatively high serum stability, can be produced in high yield andpurity (e.g., free of aggregated precursor). See, e.g., FIGS. 32A-32B.Additionally, as this nanoparticle functionalization approach is highlymodular, any desired ratio of payload, targeting ligand, or otherwise,can be introduced to the nanoparticle. Examples of preparingnanoparticles using these methods, and their benefits, are provided inthe Examples.

The NDCs of the present disclosure may comprise a structure of Formula(NP):

wherein x is an integer of 0 to 20, e.g., 4; wherein the silicon atom isa part of the nanoparticle; and wherein the

adjacent to the triazole moiety denotes a point of attachment to atargeting ligand or payload-linker conjugate, either directly orindirectly, e.g., via a linker or spacer group, e.g., a PEG moiety. Forexample, the attachment may be to a linker or spacer group, e.g., thelinker of a linker-payload conjugate, or a linker or spacer group of afolate receptor targeting ligand, e.g., a PEG moiety. The NDCs of thepresent disclosure may be prepared from diene (e.g., cyclopentadiene)functionalized nanoparticles, e.g., by conjugating a linker moiety(e.g., a linker comprising a dienophile, such as maleimide) to the dienewith a cycloaddition reaction.

The surface, e.g., silica shell surface, of a nanoparticle can bemodified by using known cross-linking agents to introduce surfacefunctional groups. Crosslinking agents include, but are not limited to,divinyl benzene, ethylene glycol dimethacrylate, trimethylol propanetrimethacrylate, N,N′-methylene-bis-acrylamide, alkyl ethers, sugars,peptides, DNA fragments, or other known functionally equivalent agents.

A carrier particle, e.g., nanoparticle, may also be conjugated to acontrast agent, such as a radionuclide, in order to permit the carrierparticle, e.g., nanoparticle, to be detectable by not only opticalimaging (such as fluorescence imaging), but also other imagingtechniques, such as positron emission tomography (PET), single photonemission computed tomography (SPECT), computerized tomography (CT), andmagnetic resonance imaging (MRI.

Carrier particles, e.g., nanoparticles, may incorporate any suitablefluorescent compound, such as a fluorescent organic compound, a dye, apigment, or a combination thereof Such fluorescent compounds can beincorporated into the silica matrix of the core of the nanoparticle. Awide variety of suitable chemically reactive fluorescentdyes/fluorophores are known, see for example, MOLECULAR PROBES HANDBOOKOF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6^(th) ed., R. P.Haugland, ed. (1996).

In preferred aspects of the present disclosure, the fluorescent compoundis covalently encapsulated within the core of the nanoparticle.

In some aspects, fluorescent compound can be, but is not limited to, anear infrared fluorescent (NIRF) dye. NIRFs may be positioned within thesilica core of a nanoparticle, that can provide greater brightness andfluorescent quantum yield relative to the free fluorescent dye. It iswell-known that the near infrared-emitting probes exhibit decreasedtissue attenuation and autofluorescence (Bums et al. “Fluorescent silicananoparticles with efficient urinary excretion for nanomedicine”, NanoLetters (2009) 9(1):442-448).

Fluorescent compounds that may be used (e.g., encapsulated by an NDC) inthe present disclosure, include, but are not limited to, Cy5, Cy5.5(also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC),tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7,fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red(sulforhodamine 101 acid chloride), LIGHTCYCLER®-Red 640,LIGHTCYCLER®-Red 705, tetramethylrhodamine (TMR), rhodamine, rhodaminederivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), therhodamine derivative JA133, Alexa Fluorescent Dyes (such as ALEXA FLUOR®488, ALEXA FLUOR® 546, ALEXA FLUOR® 633, ALEXA FLUOR® 555, and ALEXAFLUOR® 647), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide,aminomethylcoumarin (AMCA), Spectrum Green, Spectrum Orange, SpectrumAqua, LISSAMINE™, and fluorescent transition metal complexes, such aseuropium.

Fluorescent compounds that can be used also include fluorescentproteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP),blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite,mKalamal), cyan fluorescent protein and derivatives (CFP, ECFP,Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP,Citrine, Venus, YPet) (WO 2008/142571, WO 2009/056282, WO 1999/22026).In preferred aspects of the present disclosure, the fluorescent compoundis selected from the group consisting of Cy5 and Cy5.5. In preferredaspects, the fluorescent compound is Cy5. In other aspects, thefluorescent compound is Cy5.5.

A fluorescent nanoparticle may be synthesized by the steps of: (1)covalently conjugating a fluorescent compound, such as a reactivefluorescent dye, with a reactive moiety including, but not limited to,maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccimide ester,4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinimidyl ester,sulfodichlorophenol esters, sulfonyl chloride, hydroxyl, isothiocyanate,carboxyl, to an organo-silane compound, such as a co-reactiveorgano-silane compound, to form a fluorescent silica precursor, andreacting the fluorescent silica precursor to form a fluorescent core; or(2) reacting the fluorescent silica precursor with a silica formingcompound, such as tetraalkoxysilane, to form a fluorescent core. Thefluorescent core may then be reacted with a silica forming compound,such as a tetraalkoxysilane, to form a silica shell on the core, toprovide the fluorescent nanoparticle.

Fluorescent silica-based nanoparticles are known in the art and aredescribed by U.S. Pat. No. 8,298,677 B2, U.S. Pat. No. 9,625,456 B2,U.S. Ser. No. 10/548,997 B2, U.S. Pat. No. 9,999,694 B2, U.S. Ser. No.10/039,847 B2 and U.S. Ser. No. 10/548,998 B2, the contents of which areeach incorporated herein by reference in their entireties.

In preferred aspects of the present disclosure, the NDCs comprise ananoparticle that comprises a silica-based core and a silica shellsurrounding at least a portion of the core and polyethylene glycol (PEG)is covalently bonded to the surface of the nanoparticle, and afluorescent compound is covalently encapsulated within the core of thenanoparticle.

Targeting Ligands

The carrier particle drug conjugates, e.g., NDCs, of the presentdisclosure may comprise a targeting ligand that is attached to thecarrier particle, e.g., nanoparticle, directly or indirectly through aspacer group. Carrier particle drug conjugates (e.g., NDCs) withtargeting ligands can enhance internalization of the payload/drugs intumor cells and/or deliver drugs into tumor cells due to increasedpermeability, as well as the targeting ability of the carrier particledrug conjugate.

The targeting ligand can allow the carrier particle, e.g., nanoparticle,to target a specific cell type through the specific binding between theligand and the cellular component. The targeting ligand may alsofacilitate entry of the carrier particle, e.g., nanoparticle, into thecell or barrier transport, for example, for assaying the intracellularenvironment.

The targeting ligands of the present disclosure are capable of bindingto receptors on tumor cells. Specifically, the targeting ligands canbind to the folate receptor (FR), including all four human isoforms ofFR, including FR alpha (FRα, also known as FOLRT), FR beta (FRO, alsoknown as FOLR2), FR gamma (FRy, also known as FOLR3), and FR delta (FR6,also known as FOLR4). Conjugation of FR targeting ligand to the surfaceof the carrier particle, e.g., nanoparticle, of the present disclosureallows for targeted therapy of FR-overexpressing cancerous cells,tissues, and tumors.

For example, carrier particle drug conjugates, e.g., NDCs, of thepresent disclosure comprising targeting ligands that can bind to folatereceptor alpha (FRα), such as folic acid, may be used for targetingovarian cancer, endometrial cancer, fallopian tube cancer, peritonealcancer, cervical cancer, breast cancer, lung cancer, mesothelioma,uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, coloncancer, rectal cancer, and stomach cancer), pancreatic cancer, bladdercancer, kidney cancer, liver cancer, head and neck cancer, brain cancer,thyroid cancer, skin cancer, prostate cancer, and testicular cancer,acute myeloid leukemia (AML, e.g., pediatric AML). Carrier particle drugconjugates, e.g., NDCs, of the present disclosure comprising targetingligands that can bind to folate receptor beta (FRO) may be used fortargeting acute myeloid leukemia (AML, e.g., pediatric AML), chronicmyelogenous leukemia (CML), and tumor associated macrophages. Tumorassociated macrophages can be targeted as a means to modify the immunestatus of the tumor. Without wishing to be bound by theory, the bindingaffinity of FR-targeted, carrier particle drug conjugates, e.g., NDCs,to folate receptors can be enhanced due to multivalence effect.

Folate receptor can be highly expressed in solid tumor cells, includingovarian, kidney, lung, brain, endometrial, colorectal, pancreatic,gastric, prostate, breast and non-small-cell lung cancers. FR isover-expressed in other cancers including fallopian tube cancer,cervical cancer, mesothelioma, uterine cancer, esophageal cancer,stomach cancer, bladder cancer, liver cancer, head and neck cancer,thyroid cancer, skin cancer, and testicular cancer, and other cancersdisclosed herein. FR is also over-expressed in hematologicalmalignancies, such as acute myeloid leukemia (AML) and chronicmyelogenous leukemia (CML).

In preferred aspects of the present disclosure, the targeting ligandsbind to folate receptor alpha (FRu), folate receptor beta (FRO), orboth.

The present disclosure provides FR-targeting ligands that are capable ofbinding to specific cell types having elevated levels of FRα, such as,but not limited to, cancer (e.g., adenocarcinomas) of uterus, ovary,breast, cervix, kidney, colon, testicles (e.g., testicularchoriocarcinoma), brain (e.g., ependymal brain tumors), malignantpleural mesothelioma, and nonfunctioning pituitary adenocarcinoma. Thepresent disclosure also provides FR-targeting ligands that are capableof targeting acute myeloid leukemia (AML, e.g., pediatric AML), chronicmyelogenous leukemia (CML), and tumor associated macrophages. Thetargeting ligand can be any suitable molecule that can bind a FR, suchas FRα, such as a small organic molecule (e.g., folate or a folateanalog), an antigen-binding portion of an antibody (e.g. a Fab fragment,a Fab′ fragment, a F(ab′)₂ fragment, a scFv fragment, a Fv fragment, adsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, or anisolated complementarity determining region (CDR) region), an antibodymimetic (e.g., aptamer, an affibody, affilin, affimer, anticalin,avimer, Darpin, and the like), a nucleic acid, lipid, and the like.

In aspects of the present disclosure, the targeting ligand is selectedfrom the group consisting of folic acid, dihydrofolic acid,tetrahydrofolic acid, and folate receptor binding derivatives of any ofthe foregoing. In certain aspects of the present disclosure, thetargeting ligand is folic acid. In other aspects of the presentdisclosure, the targeting ligand is dihydrofolic acid. In other aspectsof the present disclosure, the targeting ligand is tetrahydrofolic acid.In other aspects of the present disclosure, the targeting ligand is afolate receptor binding derivative of any of the foregoing. It will beunderstood that “folic acid,” “dihydrofolic acid,” “tetrahydrofolicacid” encompass any amide or ester derivative of folic acid,dihydrofolic acid, and tetrahydrofolic acid, respectively. For example,free folic acid, free dihydrofolic acid, free tetrahydrofolic acid, orany folate receptor binding derivatives thereof, may be modified to beconjugated to the nanoparticle via a spacer group, such as PEG or a PEGderivative (e.g., by forming an amide bond between the terminalcarboxylic acid of the folic acid, dihydrofolic acid, tetrahydrofolicacid, or folate receptor binding derivatives thereof, and a nitrogenatom of the spacer group).

In another aspect of the present disclosure, the folate-receptortargeting ligand is a macromolecule, such as a protein, a peptide, anaptamer, an antibody, or an antibody fragment that can target a folatereceptor. For example, the folate-receptor targeting ligand may includea portion of an intact antibody, such as, for example, theantigen-binding or variable region of an antibody. Examples offolate-receptor targeting antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a scFvfragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment,a Fd fragment, or an isolated complementarity determining region (CDR)region. An antigen binding fragment of an antibody may be produced byany means. For example, an antigen binding fragment of an antibody maybe enzymatically or chemically produced by fragmentation of an intactantibody and/or it may be recombinantly produced from a gene encodingthe partial antibody sequence. Alternatively or additionally, antigenbinding fragment of an antibody may be wholly or partially syntheticallyproduced.

Folate receptor (FR)-targeted NDCs, may not only accumulate in a cancercell or tumor, but may also penetrate the tumor tissue and deliverpayloads to the entire tumor tissue for optimal treatment efficacy.Without wishing to be bound by any particular theory or mechanism, it isbelieved that the targeting ligands bind to the specific receptor groupson the surface of the cancer cell, resulting in receptor-mediated celluptake of NDCs. This receptor-mediated cell uptake of NDCs happens viathe endocytosis process, and eventually traffics NDCs to endosomes andlysosomes in cancer cells.

In aspects of the present disclosure, the carrier particle drugconjugate, eg., NDC, comprises a targeting ligand that is attached tothe carrier particle, e.g., nanoparticle, directly or indirectly througha spacer group. For example, the targeting ligand can be attached to ananoparticle directly via the silica surface of the nanoparticle (i.e.,covalently bonded). In preferred aspects, the targeting ligand isattached to the nanoparticle indirectly through a suitable spacer group.

The spacer group can be any group that can act as a spacer, e.g., as aspacer between a targeting ligand and the carrier particle, e.g.,nanoparticle, and attach the targeting ligand to the carrier particle,e.g., nanoparticle. The spacer group may be a divalent linker, such as adivalent linker that comprises a chain length of between about 5 andabout 200 atoms (e.g., carbon atoms, heteroatoms, or a combinationthereof), such as between about 5 and about 100 atoms, about 5 and about80 atoms, between about 10 and about 80 atoms, between about 10 andabout 70 atoms, between about 10 and about 30 atoms, between about 20and about 30 atoms, between about 30 and about 80 atoms, or betweenabout 30 and about 60 atoms. Suitable spacer groups may comprise analkylene, alkenylene, alkynylene, heteroalkylene (e.g., PEG),carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combination thereofFor example, the spacer group may comprise a PEG group, an alkylenegroup, or a combination thereof. The spacer group may be substituted orunsubstituted, e.g., the spacer group may comprise a substitutedalkylene, substituted heteroalkylene, or a combination thereof Forexample, the spacer group may comprise a PEG group (or PEG spacer), analkylene group (or alkylene spacer), one or more heteroatoms, and/or oneor more cyclic groups (e.g., heterocyclylene groups, such as apiperazine). For example, the targeting ligand-spacer group intermediate(TL-I) possesses a spacer group comprising a PEG group and aheterocyclylene (piperazine) moiety:

The targeting ligand, such as folic acid, may be attached to the carrierparticle, e.g., nanoparticle, indirectly through a PEG spacer group. Forexample, folic acid may be present in an NDC as an amide, e.g., tofacilitate conjugation to a PEG spacer group or other divalent linker,e.g., as shown in FIG. 1 and in the structure of (TL-I) above. Thenumber of PEG monomers in a PEG spacer may range from 2 to 20, from 2 to10, from 2 to 8, or from 2 to 5. In preferred aspects, the number of PEGgroups as spacers in a functionalized FR-targeting ligand is 3.

The average nanoparticle to targeting ligand ratio may range from about1 to about 50, from about 1 to about 40, from about 1 to about 30, orfrom about 1 to about 20. For example, the average nanoparticle totargeting ligand ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40,or 1:50. For example, the average nanoparticle to targeting ligand ratiomay range from about 1 to about 20, e.g., the average number oftargeting ligands on each nanoparticle may be between about 5 and about10, between about 10 and about 15, or between about 15 and about 20,e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14, orabout 15 targeting ligands per nanoparticle. An NDC disclosed herein maycomprise about 10 targeting ligands. An NDC disclosed herein maycomprise about 11 targeting ligands. An NDC disclosed herein maycomprise about 12 targeting ligands. An NDC disclosed herein maycomprise about 13 targeting ligands. An NDC disclosed herein maycomprise about 14 targeting ligands. An NDC disclosed herein maycomprise about 15 targeting ligands.

A smaller the number of targeting ligands attached to the nanoparticlemay help maintain the hydrodynamic diameter of the nanoparticle, e.g.,to meet the renal clearance cutoff size range (Hilderbrand et al.,Near-infrared fluorescence: Application to in vivo molecular imaging,Curr. Opin. Chem. Biol. (2010) 14:71-9). The number of targeting ligandsmeasured may be an average number of targeting ligands attached to morethan one nanoparticle. Alternatively, one nanoparticle may be measuredto determine the number of targeting ligands attached.

The number of targeting ligands attached to the nanoparticle can bemeasured by any suitable methods, such as, but not limited to, opticalimaging, fluorescence correlation spectroscopy (FCS), UV-Vis,chromatography, mass spectroscopy, or indirect enzymatic analysis.

The targeting ligand can be attached to a carrier particle, e.g.,nanoparticle, via covalent bonding to the carrier particle surface,e.g., the silica surface of a nanoparticle. Attachment can be directly,or indirectly through a spacer group. The ligand may be conjugated to acarrier particle, e.g., nanoparticle, via a functional group on thecarrier particle surface, for example, using coupling reactions, such asClick Chemistry (e.g., a 3+2 Click Chemistry reaction), cycloaddition(e.g., a 3+2 or 2+4 cycloaddition reaction, using the appropriatefunctional groups), or conjugation via a carboxylate, ester, alcohol,carbamide, aldehyde, amine, sulfur oxide, nitrile oxide, nitrone,nitrogen oxide, halide, or any other suitable compound known in the art.

In preferred aspects of the present disclosure, the conjugation ofFR-targeting ligands can be accomplished by “click chemistry” reactionusing a diarylcyclooctyne (DBCO) group. Any suitable reaction mechanismmay be adapted in the present disclosure for “click chemistry”, so longas facile and controlled attachment of the targeting ligand to thecarrier particle, e.g., nanoparticle, can be achieved.

In some aspects, a free triple bond (e.g., terminal alkyne) isintroduced onto the surface of a carrier particle, e.g., nanoparticle,e.g., via a PEG covalently conjugated with the shell of thenanoparticle, or through another suitable linker or spacer group.Separately, an azide group, or other group that is reactive with atriple bond, may be introduced onto the desired targeting ligand. Forexample, a targeting ligand (e.g., folic acid) may be modified byconjugating a group on the targeting ligand (e.g., a terminal carboxylicacid) with a spacer group (e.g., a PEG moiety) that comprises an azideat one terminus. The carrier particle (e.g., PEGylated nanoparticle)comprising the free triple bond, and the targeting ligand (comprising agroup reactive with the triple bond), can be mixed (with or without acopper or other metal catalyst) to effect cycloaddition of the groupreactive with the triple bond (e.g, azide) to the triple bond, resultingin the conjugation of the targeting ligand with the carrier particle,e.g., nanoparticle, such as using “Click Chemistry”). Many variations ofthis approach can also be used, as will be readily apparent to a personof ordinary skill in the art.

In other aspects, a maleimide functional group and a thiol group may beintroduced onto the carrier particle, e.g., nanoparticle, and thedesired targeting ligand, with the carrier particle, e.g., nanoparticle,having the maleimide functional group, the targeting ligand having thethiol group, or vice versa. The double bond of maleimide readily reactswith the thiol group to form a stable carbon-sulfur bond.

In yet another aspect, an activated ester functional group, such as asuccinimidyl ester group, and an amine group may be introduced onto thecarrier particle, e.g., nanoparticle, and the targeting ligand. Theactivated ester group readily reacts with the amine group to form astable carbon-nitrogen amide bond.

In some aspects, the FR-targeting ligand may be functionalized with asuitable terminal group to facilitate a 3+2 cycloaddition reaction, suchas nitrile oxide or nitrone group.

An azide functionalized FR-ligand (where the FR-ligand may comprise aspacer group, and the spacer group may possess the azide group) can beattached to a carrier particle, e.g., nanoparticle, either directly orindirectly via an alkyne (e.g., DBCO group). Spacer groups, such as, butnot limited to PEG groups, can be present in a FR-targeting ligandprecursor, and may possess a terminal group (e.g., azide) to facilitateconjugation to a carrier particle, e.g., nanoparticle, and afterconjugation, the spacer group may be disposed between the targetingligand and the carrier particle, e.g., nanoparticle. For example, theFR-targeting ligand precursor may comprise a structure of Formula (D-1):

wherein y is an integer of 0 to 20 (e.g., 3). For example, y may be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,e.g., 2, 3, or 4.

In some aspects, the FR-targeting ligand may be functionalized with asuitable terminal group, such as, but not limited to an azide group. Theazide functionalized FR-ligand can be attached to a carrier particle,e.g., nanoparticle, either directly or indirectly via an alkyne, such asa DBCO group on the carrier particle. Spacer groups, such as, but notlimited to PEG groups can be present between the azide functionalizedFR-ligand and the carrier particle, e.g., nanoparticle.

In preferred aspects, the FR-targeting ligand is functionalized toinclude spacer groups, such as, but not limited to PEG groups thatterminate with an azide group that can react with a DBCO group on thesurface of a carrier particle, e.g., nanoparticle.

The functionalization of FR-targeting ligand may include hydrophilic PEGgroups as spacers in order to enhance solubility in water and reduces oreliminates aggregation and precipitation problems.

In aspects of the present disclosure, the number of PEG groups asspacers that can be present in a functionalized FR-targeting ligand maybe in the range of from 2 to 20, from 2 to 10, from 2 to 8, or from 2 to5. In preferred aspects, the number of PEG groups as spacers in afunctionalized FR-targeting ligand is 3.

The NDCs of the present disclosure comprising a targeting ligand maycomprise a structure of Formula (NP-2′):

wherein T is a targeting ligand disclosed herein; x is an integer of 0to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, e.g., 4), and y is aninteger of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20, e.g., 3), and the silicon atom is a part ofthe nanoparticle (e.g., bonded with the silica shell of a core-shellsilica nanoparticle). For example, x may be 4, and y may be 3.

The NDCs of the present disclosure comprising a targeting ligand maycomprise a structure of Formula (NP-2):

wherein x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, e.g., 4), and y is an integer of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 3), and thesilicon atom is a part of the nanoparticle (e.g., bonded with the silicashell of a core-shell silica nanoparticle). For example, x may be 4, andy may be 3.

Each nanoparticle of the NDCs disclosed herein may comprise more thanone molecule of Formula (NP-2′), for example, the nanoparticle maycomprise between about 1 and about 20 molecules of Formula (NP-2′),e.g., between about 5 and about 20 molecules of Formula (NP-2′), betweenabout 8 and about 15 molecules of Formula (NP-2′), between about 10 andabout 15 molecules of Formula (NP-2′), e.g., about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, or about 20 molecules of Formula (NP-2′). An NDC disclosedherein may comprise about 12 molecules of Formula (NP-2′). An NDCdisclosed herein may comprise about 13 molecules of Formula (NP-2′).

Each nanoparticle of the NDCs disclosed herein may comprise more thanone molecule of Formula (NP-2), for example, the nanoparticle maycomprise between about 1 and about 20 molecules of Formula (NP-2), e.g.,between about 5 and about 20 molecules of Formula (NP-2), between about8 and about 15 molecules of Formula (NP-2), between about 10 and about15 molecules of Formula (NP-2), e.g., about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, or about 20 molecules of Formula (NP-2). An NDC disclosedherein may comprise about 12 molecules of Formula (NP-2). An NDCdisclosed herein may comprise about 13 molecules of Formula (NP-2).

Linker-Payload Conjugates

Disclosed herein are payloads that are suitable for conjugation to acarrier particle (e.g., nanoparticle). The payloads may be conjugated toa carrier particle (e.g., nanoparticle) by a linker (i.e., as alinker-payload conjugate). Any suitable payload can be conjugated to alinker disclosed herein. Exemplary payloads that can be conjugated tothe linkers in the present invention are cytotoxic drugs, particularlythose which are used for cancer therapy. Such drugs include, in general,DNA damaging agents, anti-metabolites, natural products, and theiranalogs. The carrier particle drug conjugates, e.g., NDCs, of thepresent disclosure can also comprise a linker-payload conjugate that isattached to the carrier particle, e.g., nanoparticle, directly orindirectly through a spacer group. In preferred aspects, thelinker-payload conjugate is attached to the carrier particle, e.g.,nanoparticle, through a spacer group.

The spacer group can be any group that can act as a spacer, e.g., as aspacer between a payload/linker conjugate and the carrier particle,e.g., nanoparticle, and attach the linker-payload conjugate to thecarrier particle, e.g., nanoparticle. The spacer group may be a divalentlinker, such as a divalent linker that comprises a chain length ofbetween about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, ora combination thereof), such as between about 5 and about 100 atoms,about 5 and about 80 atoms, between about 10 and about 80 atoms, betweenabout 10 and about 70 atoms, between about 10 and about 30 atoms,between about 20 and about 30 atoms, between about 30 and about 80atoms, or between about 30 and about 60 atoms. Suitable spacer groupsmay comprise an alkylene, alkenylene, alkynylene, heteroalkylene (e.g.,PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combinationthereof For example, the spacer group may comprise a PEG group, analkylene group, or a combination thereof. The spacer group may besubstituted or unsubstituted, e.g., the spacer group may comprise asubstituted alkylene, substituted heteroalkylene, or a combinationthereof For example, the spacer group may comprise a PEG group (or PEGspacer), an alkylene group (or alkylene spacer), one or moreheteroatoms, and/or one or more or cyclic groups.

Any desirable payload can be conjugated to a carrier particle disclosedherein (e.g., a nanoparticle), which may be via a linker, such as via alinker disclosed herein). Exemplary drugs that can be conjugated to acarrier particle include cytotoxic drugs, particularly those which areused for cancer therapy. Such drugs include, in general, DNA damagingagents, anti-metabolites, natural products, and their analogs.

Exemplary classes of cytotoxic agents include enzyme inhibitors (see fore.g., “A Review of Evaluation of Enzyme Inhibitors in Drug Discovery,”Robert A. Copeland, John Wiley and Sons, Hoboken, N.J.), such astopoisomerase inhibitors (e.g., exatecan, SN-38, topotecan, irinotecan,camptothecin, belotecan, indenoisoquinoline, phenanthridines,indolocarbazoles, and analogs thereof), dihydrofolate reductaseinhibitors, thymidylate synthase inhibitors, DNA intercalators, DNAminor groove binders, tubulin disruptors, DNA cleavers, anthracyclines,vinca drugs, mitomycins (e.g., mitomycin-C, mitomycin-A), bleomycins,cytotoxic nucleosides, pteridines, diynenes, enediyne, podophyllotoxins,dolastatins, auristatins (e.g., monomethyl auristatin E (MMAE),monomethyl auristatin F (MMAF)), maytansinoids, differentiationinducers, duocarmycin, and taxanes (e.g., taxol). For example, thepayload of an NDC disclosed herein may be exatecan, SN-38, topotecan,irinotecan, belotecan, 9-amino camptothecin, etoposide, camptothecin,taxol, esperamicin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one,podophyllotoxin, anguidine, vincristine, vinblastine, duocarmycin, apyrrolobenzodiazepine, morpholine-doxorubicin, N-(5,5-diacetoxy-pentyl)doxorubicin, a compound described in U.S. Pat. No. 5,198,560,daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin,N8-acetyl spermidine, a pyrrolobenzodiazepine, 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabine,a dolastatin, an auristatins, a calicheamicin hydrazide, esperamicin and6-mercaptopurine, methotrexate, butyric acid, retinoic acid or aderivative thereof, or a combination of any of the foregoing.

It will be understood that chemical modifications may be made to thedesired payload in order to make reactions of the payload with linkermore convenient for purposes of preparing conjugates of the presentdisclosure. For example a functional group, e.g., amine, hydroxyl, orsulfhydryl, may be appended to the payload (e.g., drug) at a positionwhich has minimal or an acceptable effect on the activity or otherproperties of the payload. Alternatively, an existing functional groupon the payload (e.g., pendant amine group) may be the point ofattachment to the linker.

For example, drugs containing an amine functional group suitable forcoupling to the linker moiety include, for example, exatecan, analogs ofexatecan, mitomycin-C, mitomycin-A, daunorubicin, doxorubicin,aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetylspermidine, pyrrolobenzodiazepines, 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabine,dolastatins (including auristatins), calicheamicin hydrazides, andderivatives thereof.

Drugs containing a hydroxyl functional group suitable for coupling tothe linker moiety include, for example, SN38, analogs of SN38,etoposide, camptothecin, taxol, esperamicin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one,podophyllotoxin, anguidine, vincristine, vinblastine, duocarmycin,pyrrolobenzodiazepines, morpholine-doxorubicin, N-(5,5-diacetoxy-pentyl)doxorubicin, compounds described in U.S. Pat. No. 5,198,560, andderivatives thereof.

Drugs containing a sulfhydryl functional group suitable for coupling tothe linker moiety include, for example, esperamicin and6-mercaptopurine, and derivatives thereof.

Drugs containing one or more carboxyl functional groups suitable forcoupling to the linker moiety include, for example, methotrexate,camptothecin (ring-opened form of the lactone), butyric acid, retinoicacid, and derivatives thereof.

In preferred aspects of the present disclosure, the payload is selectedfrom a group consisting of dihydrofolate reductase inhibitors,thymidylate synthase inhibitors and topoisomerase (Topo-1) inhibitors.

The payload of a carrier particle drug conjugate disclosed herein, e.g.,an NDC, such as a Topo-1 inhibitor payload, can be cleaved from thecarrier particle, e.g., nanoparticle, upon exposure to a chemicalenvironment inside a cell or cell organelle, e.g., upon contact with anenzymes, a low pH, or a redox-sensitive condition, thereby releasing thepayload (e.g., the Topo-1 inhibitor) inside the targeted cell (e.g.,cancer cell). Topo-1 inhibitors can stabilize the complexes of DNA andTopo-1 enzyme, preventing DNA relegation and inducing lethal DNA strandbreaks. The generation of these DNA lesions is effective for killingcancer cells, allowing carrier particle drug conjugates, e.g., NDCs, ofthe present disclosure to achieve the desired therapeutic effect.

In an NDC of the present disclosure, the payload may be any suitablecytotoxic drug, such as a cytotoxic drug disclosed herein. For example,the payload can be a topoisomerase inhibitor, such as a topoisomeraseinhibitor is selected from a group consisting of SN38, analogs of SN38,exatecan and analogs of exatecan, or salts thereof. In an aspect of thepresent disclosure, the payload is SN38, or a salt thereof. In anotheraspects of the present disclosure, the payload is an analog of SN38, ora salt thereof. In another aspect of the present disclosure, the payloadis exatecan, or a salt thereof. In yet another aspect of the presentdisclosure, the payload is an analog of exatecan, or a salt thereof.

In aspects of the present disclosure, the average nanoparticle topayload ratio ranges from 1 to 80, from 1 to 70, from 1 to 60, from 1 to50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 15, from 1 to 12and preferably from 1 to 10. For example, the average nanoparticle topayload ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21,1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36,1:38, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or 1:80. Forexample, the average number of payloads on each nanoparticle may bebetween about 5 and about 10, between about 10 and about 15, betweenabout 15 and about 20, between about 20 and about 25, or between about25 and about 30, e.g., about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, or about 30 payload molecules per nanoparticle.An NDC disclosed herein may comprise about 18 payload molecules. An NDCdisclosed herein may comprise about 19 payload molecules. An NDCdisclosed herein may comprise about 20 payload molecules. An NDCdisclosed herein may comprise about 21 payload molecules. An NDCdisclosed herein may comprise about 22 payload molecules. An NDCdisclosed herein may comprise about 23 payload molecules. An NDCdisclosed herein may comprise about 24 payload molecules. An NDCdisclosed herein may comprise about 25 payload molecules. An NDCdisclosed herein may comprise about 26 payload molecules. An NDCdisclosed herein may comprise about 27 payload molecules.

Vintafolide, developed by Endocyte and Merck & Co. is a small moleculedrug conjugate consisting of a small molecule targeting the FolateReceptor, which is over expressed on certain cancers, such as ovariancancer, and a chemotherapy drug, Vinblastine (U.S. Pat. No. 7,601,332 B2and U.S. Pat. No. 1,002,942 B2). However, vintafolide is capable ofcarrying single molecule of payload only, attached to the targetingmoiety by a pH-cleavable linker. In contrast to that, in the presentdisclosure several cytotoxic payload can be incorporated onto thesurface of single nanoparticle.

The linkers in the linker-payload conjugates may be self-immolativelinkers that are capable of releasing the active payload in vitro aswell as in vivo under various conditions such as, but not limited to,conditions sufficient for enzymatic release of the active payload (e.g.,a condition presenting an enzyme capable of catalyzing the release);conditions sufficient for reduction or oxidation (redox) to effect therelease of the payload (e.g., a condition presenting a substance capableof a reduction or oxidation reaction (redox) to effect the release ofthe active payload), or an acidic or basic condition sufficient toeffect the release of the payload (e.g., a condition presenting asubstance sufficient to lower or raise pH and effect release of theactive payload).

The linkers described herein can be used, for example, to attach acytotoxic drug payload to a carrier and/or a targeting moiety that bindsto a cancer cell (e.g., binds to a receptor on the surface of a cancercell) and gets internalized into the cell (e.g., through the endosomeand lysosomal compartment). Once internalized the linkers can be cleavedor degraded to release active cytotoxic drug. The pH sensitive linkerscan release payloads under acidic conditions, the redox sensitivelinkers may release payloads under the action of glutathione, which ispresent in the lysosome or endosome; and the protease-cleavable linkerscan release their payload under the action of proteases such ascathepsin, trypsin or other proteases in the lysosomal compartment ofthe cell.

The cleavable linkers described herein may comprise a structure ofFormula (F):

wherein each instance of [AA] is a natural or non-natural amino acidresidue; z is an integer of 1 to 5; w is an integer of 1 to 4 (e.g., 2or 3); and each

denotes a point of attachment, e.g., to a spacer group (e.g., PEG) oranother portion of the linker, or to a payload molecule. For example,-[AA]_(w)- may comprise Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys,Val-Arg, or Val-Ala, and z may be 2, wherein one

denotes an attachment to the oxygen atom of a PEG group, and the other

denotes an attachment to a payload. For example, -[AA]_(w)- may compriseVal-Lys.

The cleavable linkers described herein may comprise a structure ofFormula (F-1):

wherein one

denotes a point of attachment to the oxygen atom of a PEG group, and theother

denotes a point of attachment to a payload.

Also disclosed herein are linker molecules (sometimes referred to hereinas linker precursors) that are useful for conjugating compounds (e.g.,payload moieties, or targeting ligand moieties) to a carrier particle(such as a nanoparticle). The linkers of this disclosure can containreactive groups at one or both ends of the molecule. The reactive groupscan be selected to allow conjugation to any desired compound, such as apayload (e.g., cytotoxic payload) at one end, and also facilitateconjugation to the carrier particle (e.g., nanoparticle) at the otherend. The desired payload (e.g., cytotoxic payload) may or may not bemodified. It is desirable for the payload to contain an amine, ahydroxyl, hydrazone, hydrazide or a sulfhydryl group in order tofacilitate conjugation to the linker.

The linker-payload conjugates (or precursors) can be attached to thecarrier particle (e.g., nanoparticle) using any suitable techniques andmethods, and many such techniques are well-known in the art. See, e.g.,WO 2017/189961, WO 2015/183882, WO 2013/192609, WO 2016/179260 and WO2018/213851, each of which are hereby incorporated by reference in theirentireties, which describe silica based core-shell or silica based corenanoparticles that can be used to prepare targeted nanoparticle-baseddrug delivery systems. Additionally, linker-payload conjugates (orprecursors), or targeting ligands (or precursors or conjugates thereof),can be attached to a carrier particle (e.g., nanoparticle) using areaction or method described in Kolb et al. Angew. Chem. Int. Ed. (2001)40:2004-2021, which is incorporated herein by reference in its entirety.

The linker-payload conjugate may be attached to the carrier particle(e.g., nanoparticle) directly or indirectly through a spacer group, suchas a spacer group described herein. Suitable spacer groups include, butare not limited to, a divalent linker (e.g., a divalent linker describedherein), such as PEG spacer, or an alkylene spacer (e.g., a methylenespacer), which may further comprise a heteroatom or cyclic group (e.g.,heterocyclylene groups). The linker-payload conjugate can be absorbedinto the interstices or pores of a silica shell, or coated onto thesilica shell of a nanoparticle, such as a fluorescent nanoparticle(e.g., covalently attached to the surface of the nanoparticle). In otheraspects, where the silica shell is not covering all of the surface ofthe nanoparticle, the linker-payload conjugate can be associated withthe fluorescent core, such as by physical absorption or by bondinginteraction.

In some aspects, the linker-payload conjugate may also be associatedwith PEG groups that are covalently bonded to the surface of a carrierparticle (e.g., nanoparticle). For example, the linker-payload conjugatemay be attached to a nanoparticle through the PEG. The PEGs can havemultiple functional groups for attachment to the nanoparticle and to thelinker-payload conjugate.

In specific aspects of the present disclosure, the linker-payloadconjugates (or linker-payload conjugate precursor) may be functionalizedwith a hydrophilic PEG spacer. The linker-payload conjugate precursormay be functionalized with a hydrophilic PEG spacer and/or suitableterminal group such as, but not limited to, an azide group, tofacilitate covalently attaching the linker-payload conjugate (e.g., viathe spacer group) to the surface of a carrier particle, such as ananoparticle, e.g., via reaction with a DBCO group on the carrierparticle surface. Other terminal groups can include a nitrile oxide ornitrone, e.g., for conjugation via a 3+2 cycloaddition reaction, to asuitable group on the nanoparticle (e.g., a diene moiety).

The number of PEG groups as spacers that can be present in afunctionalized linker-payload conjugate (or precursor thereof) may rangefrom 0 to 20, e.g., from 2 to 20, from 2 to 10, or from 5 to 8, e.g., 5,6, 7, 8, 9, 10, 11, or 12. In preferred aspects, the number of PEGgroups as spacers in a functionalized linker-payload conjugate is 9.

For example, exatecan can be conjugated to a protease-cleavable linkerto form the linker-payload conjugate. This linker-conjugate can beprepared from a precursor functionalized with a PEG spacer that has aterminal reactive group, such as an azide group, for further conjugationto the surface of the nanoparticle, e.g., via a DBCO group.

The protease-cleavable linker can be designed to be labile tocathepsin-B (Cat-B), that is over-expressed in malignant tumors, therebyeffecting release of the cytotoxic agent, such as exatecan by aself-immolative process.

The linker payload conjugate precursor can comprise a structure ofFormula (E-1′):

wherein P is a payload moiety, e.g., a cytotoxic drug described herein,and y is an integer of 0 to 20, e.g., 5 to 15, e.g., 9.

The linker and linker-payload conjugates described in the presentdisclosure have several advantages, ranging from superior serumstability to faster release kinetics mechanism, relative to conventionaldrug delivery platforms, linkers, or linker-payload conjugates. Also,the ability to pair these linkers with a variety of chemical groupsprovides the opportunity for the selective release of freepayload/drugs, with minimal derivatization, that is a significantadvantage.

In aspects of the present disclosure, the linker in the linker-payloadconjugate is selected from a group consisting of protease-cleavablelinker, redox-sensitive linker and pH-sensitive linker. In an aspect ofthe present disclosure, the linker in the linker-payload conjugate is aprotease-cleavable linker. In other aspects of the present disclosure,the linker in the linker-payload conjugate is a redox-sensitive linker.In other aspects of the present disclosure, the linker in thelinker-payload conjugate is a pH-sensitive linker.

A carrier particle-drug conjugate (e.g., an NDC) disclosed herein maycomprise more than one linker-payload conjugate moiety (e.g., alinker-payload conjugate moiety disclosed herein). For example, thecarrier particle-drug conjugate may comprise between about 1 and about80 linker-payload conjugate moieties, e.g., between about 1 and about60, between about 1 and about 40, between about 1 and about, betweenabout 10 and about 30, between about 15 and about 25 linker-payloadconjugate moieties, e.g., about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, or about 30 linker-payload conjugate moieties.

A carrier particle-drug conjugate (e.g., an NDC) disclosed herein maycomprise both a targeting ligand moiety, and a linker-payload conjugatemoiety, e.g., each carrier particle-drug conjugate may comprise about 1and about 20 targeting ligand moieties, and about 1 and about 30linker-payload conjugate moieties. For example, each carrierparticle-drug conjugate may comprise about 10 and about 15 targetingligand moieties, and about 15 and about 25 linker-payload conjugatemoieties. A carrier particle-drug conjugate disclosed herein maycomprise an average of 13 targeting ligand moieties, and an average of21 linker-payload conjugate moieties; an average of 12 targeting ligandmoieties, and an average of 25 linker-payload conjugate moieties; anaverage of 12 targeting ligand moieties, and an average of 20linker-payload conjugate moieties.

This disclosure provides compositions and methods directed to ananoparticle-drug conjugate (NDC) comprising: a nanoparticle; atargeting ligand that binds to folate receptor; and a linker-payloadconjugate, wherein the NDC has an average diameter between about 1 nmand about 10 nm.

FIG. 1 illustrates a representative nanoparticle-drug conjugate (NDC)that has an average diameter of about 6 nm, comprising a nanoparticlethat comprises a silica-based core and a silica shell surrounding atleast a portion of the core, polyethylene glycol (PEG) covalently bondedto the surface of the nanoparticle, and a fluorescent compound (Cy5)covalently encapsulated within the core of the nanoparticle, folic acid(FA) as the targeting ligand that can bind to folate receptor, and alinker-payload conjugate that comprises a protease-cleavablelinker-exatecan conjugate. It will be understood that “folic acid” isintended to encompass any amide or ester derivative of folic acid, e.g.,as shown in FIG. 1 where folic acid is covalently attached to the spacergroup (PEG) via an amide group.

In an aspect, the NDC has an average diameter between about 5 nm toabout 8 nm. In more preferred aspects of the present disclosure, the NDChas an average diameter between about 6 nm to about 7 nm. The averagediameter of NDCs can be measured by any suitable methods, such as, butnot limited to, fluorescence correlation spectroscopy (FCS) (see, e.g.,FIG. 6 ) and gel permeation chromatography (GPC) (see, e.g., FIG. 7 ).The NDCs of the present disclosure can comprise nanoparticles that canbe functionalized with contrast agents for positron emission tomography(PET), single photon emission computed tomography (SPECT), computerizedtomography (CT), magnetic resonance imaging (MRI), and optical imaging(such as fluorescence imaging including near-infrared fluorescence(NIRF) imaging, bio luminescence imaging, or combinations thereof).

A contrast agent, such as a radionuclide (radiolabel) including, but notlimited to ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu, may be attached to thenanoparticle. Alternatively, the nanoparticle can be attached to achelator moiety, for example, DFO, DOTA, TETA and DTPA, that is adaptedto bind a radionuclide. Such nanoparticle may be detected by PET, SPECT,CT, MRI, or optical imaging (such as fluorescence imaging includingnear-infrared fluorescence (NIRF) imaging, bio luminescence imaging, orcombinations thereof).

The radionuclide can additionally serve as a therapeutic agent forcreating a multitherapeutic platform. This coupling allows thetherapeutic agent to be delivered to the specific cell type through thespecific binding between the targeting ligand and the cellularcomponent.

Linkers

This disclosure provides linkers, e.g., self-immolative linkers, andlinker-drug conjugates. The self-immolative linkers are capable ofreleasing payloads (e.g., a payload described herein) in vitro as wellas in vivo under various conditions, that may involve enzymaticcleavage, redox-promoted cleavage, or pH-promoted cleavage. The linkersdescribed herein can be used, for example, to attach a payload (e.g.,cytotoxic drug) to a carrier particle and/or a targeting moiety thatbinds to a cancer cell (e.g., binds to a receptor on the surface of acancer cell) and can be internalized into the cell (e.g., through theendosome and lysosomal compartment). The payload can be any suitablepayload, e.g., a payload disclosed herein. Once internalized, thelinkers can be cleaved or degraded to release active cytotoxic drug. ThepH sensitive linkers can release payloads under acidic conditions; theredox sensitive linkers release payloads under the action of anothercompound, for example, glutathione, which may be present in the lysosomeor endosome; and protease-cleavable linkers can release a payload underthe action of an enzyme such as a protease, e.g., cathepsin, trypsin, orother proteases in the lysosomal compartment of a cell.

The linkers of this disclosure can contain reactive groups at both endsof the molecule. The reactive groups can be selected to allowconjugation to any desired cytotoxic payload at one end, and alsofacilitate conjugation to any drug-delivery system (i.e., carrierparticle) at the other end. The desired cytotoxic payload may or may notbe modified. It is desirable for the payload to contain an amine, ahydroxyl, or a sulfydryl group in order to facilitate conjugation to thelinker or linker-drug-delivery system.

The drug-delivery systems that are envisaged by this disclosure include,but are not limited to, a nanoparticle, a liposome, a nanogel, ananoring, a nanocage, a microsphere, an antibody, or antigen-bindingportion of an antibody including single chain antibodies and fragments(e.g., scFv), and the like. The drug-delivery system may be an antibodyfragment, e.g., Fab, F(ab′)₂, scFv, minibody, or nanobody. Theconjugation of reactive groups is carried out using any suitabletechniques and methods, such as a technique described herein.

This disclosure relates to linker-drug conjugates of Formulae (I)-(XII)and (I-B)-(XII-B). In some aspects, the present disclosure relates toNDCs that comprise linker-payload conjugates of Formulae (I)-(XII) and(I-B)-(XII-B). The self-immolative linkers of the present disclosure canbe conjugated to a cytotoxic agent (e.g., drug moiety or payload) suchas SN38, analogs of SN38, exatecan or analogs of exatecan, or anotherpayload disclosed herein. The linker-payload conjugates can include asub-structure of Formulae (I)-(XII) that can be conjugated eitherdirectly or indirectly to the carrier particle (e.g., nanoparticle). Inpreferred aspects of the present disclosure, the linker-payloadconjugates are conjugated indirectly to a carrier particle (e.g.,nanoparticle) through suitable spacer units, such as, but not limited toPEG spacers.

The NDCs of the present disclosure may comprise multiple differentpayloads, e.g., multiple different drugs, that may be linked to thecarrier particle (e.g., nanoparticle) via a self-immolative linker),e.g., two, three, four, five, six, seven, eight, nine, ten, or moredifferent drug molecules per nanoparticle.

Protease-Cleavable Linker-Payload Conjugates: In certain aspects of thepresent disclosure, the linker-payload conjugates of Formulae (I)-(IV)or (I-B)-(IV-B) relate to protease-cleavable linkers that are conjugatedto a cytotoxic agent (drug moiety or payload) such as SN38, analogs ofSN38, exatecan and analogs of exatecan.

A linker-payload conjugate may comprise a compound of Formula (I)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g.,

nanoparticle) or an indirect bond to the carrier particle (e.g.,nanoparticle) through a spacer group; A is a dipeptide selected from thegroup consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys,Val-Arg, and Val-Ala; or A is a tetrapeptide selected from the groupconsisting of Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar,Val-Lys-Gly-Sar (SEQ ID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11),Val-Phe-Gly-Pro (SEQ ID NO: 12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQID NO: 14), Val-Ala-Gly-Pro (SEQ ID NO: 15), Val-Cit-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Val-Phe-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Phe-Lys-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z; R¹ and R² in eachoccurrence is independently hydrogen, substituted or unsubstituted C₁₋₆alkyl or substituted or unsubstituted C₁₋₆ alkoxy, or hydroxyl; R³ andR⁴ in each occurrence is independently hydrogen, halo, substituted orunsubstituted C₁₋₆ alkyl, or substituted or unsubstituted C₁₋₆ alkoxy;R⁵ is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted C₅₋₆ heterocycloalkyl; withthe proviso that, when A is a dipeptide, R⁵ is H; R^(a), R^(b), andR^(c) in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; X is absent, —O—, —CO— or —NR^(a)—; Y isabsent

wherein the carbonyl in

is bonded to Z; with the proviso that, when Y is

X is absent and n is 1; when Y is

X is absent and n is 0; when Y is

X is absent and n is 0; and/or when X is —CO—, Y is absent and n is 0;X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is—NR^(C)— or —O—; n is 0 or 1; and q is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (I-B)

or a salt thereof, wherein: A is a dipeptide selected from the groupconsisting of Val-Cit, Phe-Lys, Trp- Lys, Asp-Lys, Val- Lys, Val-Arg,and Val-Ala; or A is a tetrapeptide selected from the group consistingof Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of a drug moiety or a pro-drug moiety, andwhen Payload is a separate molecular entity it contains an amino orhydroxyl group that provides the nitrogen or oxygen atom at; R¹ and R²in each occurrence is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy, orhydroxyl; R³ and r⁴ in each occurrence is independently hydrogen, halo,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy; R⁵ is selected from the group consisting of hydrogen,substituted or unsubstituted C₁₋₆ alkyl; substituted or unsubstitutedC₃₋₇ cycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and substituted or unsubstituted C₅₋₆heterocycloalkyl; with the proviso that, when A is a dipeptide, R⁵ is H;R^(1′), R^(2′), R^(3′), R⁴′ and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl; R^(a), R^(b) and R^(C) ineach occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; T₁ is a functionalized polyethylene glycol ora C₅-C₆ alkyl chain that has a terminal group selected from the groupconsisting of azide,

X is absent, —O—, —CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z; with the proviso that, when Y is

X is absent and n is 1; when Y is

X is absent and n is 0; when Y is

X is absent and n is 0; and/or when X is —CO—, Y is absent and n is 0;X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is—NR^(c)— or —O—; n is 0 or 1; and q is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (II)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; A is a dipeptide selected from the group consisting ofVal-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or Ais a tetrapeptide selected from the group consisting of Val-Phe-Gly-Sar(SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; Payload is aresidue of cytotoxic moiety, and when Payload is a separate molecularentity it contains an amino or hydroxyl group that provides the nitrogenor oxygen atom at Z; R¹ is hydrogen or substituted or unsubstituted C₁₋₆alkyl; R³ and R⁴ in each occurrence is independently hydrogen, halo,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy; R⁵ is selected from the group consisting of hydrogen,substituted or unsubstituted C₁₋₆ alkyl; substituted or unsubstitutedC₃₋₇ cycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and substituted or unsubstituted C₅₋₆heterocycloalkyl; with the proviso that, when A is a dipeptide, R⁵ is H;R^(a), R^(b) and R^(c) in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; X₁ and X₂ are independently—CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is

and Z is —NR^(c)— or —O—.

A linker-payload conjugate may comprise a compound of Formula (II-B)

or a salt thereof, wherein: A is a dipeptide selected from the groupconsisting of Val-Cit, Phe-Lys, Trp- Lys, Asp-Lys, Val- Lys, Val-Arg,and Val-Ala, or A is a tetrapeptide selected from the group consistingof Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of a drug moiety or a pro-drug moiety, andwhen Payload is a separate molecular entity it contains an amino orhydroxyl group that provides the nitrogen or oxygen atom at Z; R¹ ishydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵ is selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅₋₆ heterocycloalkyl; with the provisothat, when A is a dipeptide, R⁵ is H; R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R^(a), R^(b) and R^(c) in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ is afunctionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is

and; Z is —NR^(c)— or —O—.

A linker-payload conjugate may comprise a compound of Formula (III)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; A is a dipeptide selected from the group consisting ofVal-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or Ais a tetrapeptide selected from the group consisting of Val-Phe-Gly-Sar(SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; Payload is aresidue of cytotoxic moiety, and when Payload is a separate molecularentity it contains an amino or hydroxyl group that provides the nitrogenor oxygen atom at Z; R³ and R⁴ in each occurrence is independentlyhydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ alkoxy; R⁵ is selected from the group consistingof hydrogen, substituted or unsubstituted C₁₋₆ alkyl; substituted orunsubstituted C₃₋₇ cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted C₅₋₆ heterocycloalkyl, with the proviso that, when A is adipeptide, R⁵ is H; R^(c) is selected from a group consisting ofhydrogen or substituted or unsubstituted C₁₋₆ alkyl; X₁ and X₂ areindependently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; and Z is —NR^(c)— or—O—.

A linker-payload conjugate may comprise a compound of Formula (III-B):

Or a salt thereof, wherein: A is a dipeptide selected from the groupconsisting of Val-Cit, Phe-Lys, Trp- Lys, Asp-Lys, Val- Lys, Val-Arg,and Val-Ala, or A is a tetrapeptide selected from the group consistingof Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of a drug moiety or a pro-drug moiety, andwhen Payload is a separate molecular entity it contains an amino orhydroxyl group that provides the nitrogen or oxygen atom at Z; R³ and R⁴in each occurrence is independently hydrogen, halo, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted C₅₋₆ heterocycloalkyl, withthe proviso that, when A is a dipeptide, R⁵ is H; R^(1′), R²′, R^(3′),R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(c) is selected from a group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH— and; Zis —NR^(c)— or —O—.

A linker-payload conjugate may comprise a compound of Formula (IV)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha through a spacer group; A is a dipeptide selected from the groupconsisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, andVal-Ala, or A is a tetrapeptide selected from the group consisting ofVal-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ IDNO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of cytotoxic moiety, and when Payload is aseparate molecular entity it contains an amino or hydroxyl group thatprovides the nitrogen or oxygen atom at Z; R^(c) is selected from agroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;and Z is —NR^(c)— or —O—.

A linker-payload conjugate may comprise a compound of Formula (IV-B)

or a salt thereof, wherein: A is a dipeptide selected from the groupconsisting of Val-Cit, Phe-Lys, Trp- Lys, Asp-Lys, Val-Lys, Val-Arg, andVal-Ala, or A is a tetrapeptide selected from the group consisting ofVal-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ IDNO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of a drug moiety or a pro-drug moiety, andwhen Payload is a separate molecular entity it contains an amino orhydroxyl group that provides the nitrogen or oxygen atom at Z; R¹′,R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl or substituted orunsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from a group consistingof hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ is afunctionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

and Z is —NR^(c)— or —O—.

In aspects of Formulae (I)-(IV),

line represents an indirect bond to a carrier particle (e.g.,nanoparticle) through a suitable spacer, such as, but not limited to PEGspacers.

In aspects of Formulae (I)-(IV) or (I-B)-(IV-B), Payload is a residue ofa drug moiety or a prodrug moiety, and when Payload is a separatemolecular entity it contains an amino or hydroxyl group that providesthe nitrogen or oxygen atom at Z.

In aspects of Formulae (I)-(IV) or (I-B)-(IV-B), Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z. In preferred aspects of Formulae (I)-(IV) or (I-B)-(IV-B),Payload is a residue of a cytotoxic moiety.

In aspects of Formulae (I)-(IV) or (I-B)-(IV-B), including thosepreferred aspects described above, A is a dipeptide or a tetrapeptide.In certain aspects of Formulae (I)-(IV) or (I-B)-(IV-B), A is adipeptide selected from the group consisting of Val-Cit, Phe-Lys,Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala. In preferred aspects ofFormulae (I)-(IV) or (I-B)-(IV-B), A is Val-Cit. In preferred aspects ofFormulae (I)-(IV) or (I-B)-(IV-B), A is Val-Lys. In preferred aspects ofFormulae (I)-(IV) or (I-B)-(IV-B), A is Phe-Lys.

In certain aspects of Formulae (I)-(IV) or (I-B)-(IV-B), including thosepreferred aspects described above, A is a tetrapeptide selected from thegroup consisting of Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar,Val-Lys-Gly-Sar (SEQ ID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11),Val-Phe-Gly-Pro (SEQ ID NO: 12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQID NO: 14), Val-Ala-Gly-Pro (SEQ ID NO: 15), Val-Cit-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Val-Phe-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Phe-Lys-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid. In preferred aspects of Formulae (I)-(IV) or(I-B)-(IV-B), A is a tetrapeptide selected from a group consisting ofVal-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ IDNO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), andVal-Ala-Gly-Pro (SEQ ID NO: 15). In preferred aspects of Formulae(I)-(IV) or (I-B)-(IV-B), A is Val-Lys-Gly-Sar (SEQ ID NO: 10). Inpreferred aspects of Formulae (I)-(IV) or (I-B)-(IV-B), A isVal-Ala-Gly-Sar (SEQ ID NO: 11). In preferred aspects of Formulae(I)-(IV) or (I-B)-(IV-B), A is Val-Phe-Gly-Pro (SEQ ID NO: 12). Inpreferred aspects of Formulae (I)-(IV) or (I-B)-(IV-B), A isVal-Cit-Gly-Pro. In preferred aspects of Formulae (I)-(IV) or(I-B)-(IV-B), A is Val-Lys-Gly-Pro (SEQ ID NO: 14). In preferred aspectsof Formulae (I)-(IV) or (I-B)-(IV-B), A is Val-Ala-Gly-Pro (SEQ ID NO:15).

In aspects of Formula (I) or (I-B), including those preferred aspectsdescribed above, R¹ and R² in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy, or hydroxy. In preferred aspects of Formula (I) or (I-B),R¹ and R² is hydrogen. In preferred aspects of Formula (I) or (I-B), R¹and R² is methyl. In preferred aspects of Formula (I) or (I-B), R¹ ishydrogen and R² is hydroxy. In preferred aspects of Formula (I) or(I-B), R¹ is hydrogen and R² is methyl. In preferred aspects of Formula(I) or (I-B), R¹ is hydroxy and R² is methyl. In aspects of Formula (II)or (II-B), including those preferred aspects described above, R¹ isselected from a group consisting of hydrogen or substituted orunsubstituted C₁₋₆ alkyl. In preferred aspects of Formula (II) or(II-B), R¹ is hydrogen. In preferred aspects of Formula (II) or (II-B),R¹ is methyl.

In aspects of Formulae (I), (II), (III), (I-B), (II-B), or (III-B),including those preferred aspects described above, R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy. In preferredaspects of Formulae (I), (II),(III), (I-B), (II-B), or (III-B), R³ andR⁴ in each occurrence is independently hydrogen, chloro, fluoro, methylor methoxy. In preferred aspects of Formulae (I), (II),(III), (I-B),(II-B), or (III-B), R³ and R⁴ is hydrogen. In preferred aspects ofFormulae (I), (II), (III), (I-B), (II-B), or (III-B), R³ and R⁴ isfluoro.

In aspects of Formulae (I), (II). (III), (I-B), (II-B), or (III-B),including those preferred aspects described above, R⁵ is selected fromthe group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅₋₆ heterocycloalkyl, with the provisothat, when A is a dipeptide, R⁵ is H. In certain aspects of Formulae(I), (II). (III) (I-B), (II-B), or (III-B), including those preferredaspects described above, R⁵ is selected from the group consisting ofhydrogen, methyl, cyclopropyl, phenyl, and a substituted phenyl. Inpreferred aspects of Formulae (I), (II), (III), (I-B), (II-B), or(III-B), R⁵ is hydrogen.

In preferred aspects of Formulae (I), (II), (III), (I-B), (II-B), or(III-B), when A is a dipeptide, R⁵ is H. In certain aspects of Formulae(I), (II), (I-B), or (II-B), including those preferred aspects describedabove, R^(a), R^(b) and R^(c) in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl. In preferredaspects of Formulae (I) (II), (I-B), or (II-B), R^(a), R^(b) and R^(c)is hydrogen. In preferred aspects of Formulae (I), (II), (I-B), or(II-B), R^(a), R^(b) and R^(c) is methyl. In preferred aspects ofFormula (II) or (II-B), R^(b) is hydrogen and R^(b) is methyl.

In aspects of Formulae (III), (IV), (III-B), or (IV-B), including thosepreferred aspects described above, R^(c) is selected from a groupconsisting of hydrogen and substituted or unsubstituted C₁₋₆ alkyl. Inpreferred aspects of Formulae (III), (IV), (III-B), or (IV-B), R^(c) ishydrogen. In preferred aspects of Formulae (III), (IV), (III-B), or(IV-B), R^(b) is methyl.

In aspects of Formula (I) or (I-B), including those preferred aspectsdescribed above, X is absent, —O—, —CO— or —NR^(a)—. In preferredaspects of Formula (I) or (I-B), X is absent. In preferred aspects ofFormula (I) or (I-B), X is —O— and n is 0. In preferred aspects ofFormula (I) or (I-B), X is —CO— and n is 0. In preferred aspects ofFormula (I) or (I-B), X is —NR^(a)—, wherein R^(a) is selected from agroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl,preferably, R^(a) is methyl.

In aspects of Formula (I) or (I-B), including those preferred aspectsdescribed above, Y is absent,

wherein the carbonyl in

is bonded to Z; with the proviso that, when Y is

X is absent and n is 1; with the proviso that, when Y is

X is absent and n is 0; wthe proviso that, when Y is

X is absent and n is 0 or 1 and with the proviso that, when X is —CO—, Yis absent and n is 0. In preferred aspects of Formula (I) or (I-B), Y isabsent. In preferred aspects of Formula (I) or (I-B), Y is

In preferred aspects of Formula (I) or (I-B), Y is

In preferred aspects of Formula (I) or (I-B), Y is

preferably Y is —CONH₂. In preferred aspects of Formula (I) or (I-B), Yis

In preferred aspects of Formula (I) or (I-B), Y is

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (I) or (I-B), Y is

wherein the carbonyl in is

bonded to Z. In preferred aspects of Formula (I) or (I-B), Y is

wherein the carbonyl in

is bonded to Z.

In aspects of Formula (II) or (II-B), including those preferred aspectsdescribed above, Y₁ is

In certain aspects of Formula (II) or (II-B), Y₁ is

preferably, Y₁ is —COOCH₃. In certain aspects of Formula (II) or (II-B),Y₁ is

preferably, Y₁ is —CONH₂.

In aspects of Formulae (I), (II),(III), (I-B), (II-B), or (III-B),including those preferred aspects described above, X₁ and X₂ areindependently —CH— or —N—; X₃ is —CH— and X₄ is —CH—. In preferredaspects of Formulae (I), (II),(III), (I-B), (II-B), or (III-B), X₁, X₂,X₃ and X₄ are each —CH—. In preferred aspects of Formulae (I),(II),(III), (I-B), (II-B), or (III-B), X₁ is —CH—; X₂ is —N—; X₃ is —CH—and X₄ is —CH—. In preferred aspects of Formulae (I), (II),(III), (I-B),(II-B), or (III-B), X₁ is —N—; X₂ is —CH—; X₃ is —CH— and X₄ is —CH—.

In aspects of Formulae (I)-(IV) or (I-B)-(IV-B), including thosepreferred aspects described above, Z is —NR^(c)— or —O—, preferably, Zis —NR^(c)—, wherein R^(c) is hydrogen or substituted or unsubstitutedC₁₋₆ alkyl, preferably, R^(c) is methyl or preferably, Z is —O—.

In aspects of Formula (I) or (I-B), including those preferred aspectsdescribed above, n is 0 or 1 and q is 1 to 3. In aspects of Formula (I)or (I-B), including those preferred aspects described above, n is 0 andq is 1 to 3. In aspects of Formula (I) or (I-B), including thosepreferred aspects described above, n is 1 and q is 1 to 3.

Redox-Sensitive Linker-Payload Conjugates: In certain aspects of thepresent disclosure, the linker-payload conjugates of Formulae (V)-(X) or(V-B)-(X-B) relate to redox-sensitive linkers that are conjugated to acytotoxic agent (drug moiety or payload) such as SN38, analogs of SN38,exatecan and analogs of exatecan.

A linker-payload conjugate may comprise a compound of Formula (V)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z; R¹, R², R⁵, R⁶ andR⁷ in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; R³ and R⁴ in each occurrence is independentlyhydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ alkoxy; R^(a), R^(b) and R^(c) in each occurrenceis independently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; Xis absent, —O—, —CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z; X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄is —CH—; Z is —NR^(c)— or —O—; n is 0 or 1; p is 1 to 3; and q is 1 to3.

A linker-payload conjugate may comprise a compound of Formula (V-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) in each occurrence isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ isa functionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

X is absent, —O—, —CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z; X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄is —CH—; Z is —NR^(c)— or —O—; n is 0 or 1; p is 1 to 3; and q is 1 to 3

A linker-payload conjugate may comprise a compound of Formula (VI)

or a salt thereof, wherein,

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha suitable spacer, such as, but not limited to PEG spacers; Payload is aresidue of cytotoxic moiety, and when Payload is a separate molecularentity it contains an amino or hydroxyl group that provides the nitrogenor oxygen atom at Z; R¹, R⁵, R⁶ and R⁷ in each occurrence isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³and R⁴ in each occurrence is independently hydrogen, halo, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy;R^(a), R^(b) and R^(c) in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; X₁ and X₂ are independently—CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is

Z is —NR— or —O— and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (VI-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or aprodrug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) in each occurrence isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ isa functionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is—; Y₁ is

Z is —NR^(c)— or —O—; and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (VII)

or a salt thereof,

wherein, line represents a direct bond to a carrier particle (e.g.,nanoparticle) or an indirect bond to the carrier particle (e.g.,nanoparticle) through a spacer group; Payload is a residue of cytotoxicmoiety, and when Payload is a separate molecular entity it contains anamino or hydroxyl group that provides the nitrogen or oxygen atom at Z;R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; Y is

wherein the carbonyl in or

is bonded to Z; Z is —NR^(c)— or —O— and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (VII-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or aprodrug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(c) is selected from a group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

wherein the carbonyl in

or is bonded to Z; Z is —NR^(c)— or —O—; and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (VIII)

or a salt thereof, wherein

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z; R¹, R², R³, R⁵, R⁶and R⁷ in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; R⁴ is halo; R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; Z is—NR^(c)— or —O—; m is 1 to 3; and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (VIII-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R³, R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R⁴ is halo; R^(1′),R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl or substituted orunsubstituted C₁₋₆ cycloalkyl; R¹ is selected from a group consisting ofhydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ is afunctionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

Z is —NR^(c)— or —O—; m is 1 to 3; and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (IX)

or a salt thereof, wherein

line represents a direct bond to a carrier particle (e.g.,

nanoparticle) or an indirect bond to the carrier particle (e.g.,nanoparticle) through a spacer group; Payload is a residue of cytotoxicmoiety, and when Payload is a separate molecular entity it contains anamino or hydroxyl group that provides the nitrogen or oxygen atom at Z;R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; Z is—NR^(c)— or —O—; m is 1 to 3 and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (IX-B)

or a salt thereof, wherein; Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R^(1′), R²′,R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(c) is selected from a group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

Z is —NR^(c)— or —O—; m is 1 to 3; and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (X)

or a salt thereof, wherein

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵, R⁶ and R⁷ ineach occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; R^(c) is selected from a group consisting ofhydrogen or substituted or unsubstituted C₁₋₆ alkyl; X₁ and X₂ areindependently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is —NR^(c)— or —O—and p is 1 to 3.

A linker-payload conjugate may comprise a compound of Formula (X-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R³ and R⁴ in each occurrence is independently hydrogen, halo,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy; R⁵, R⁶ and R⁷ in each occurrence is independently hydrogenor substituted or unsubstituted C₁₋₆ alkyl; R^(1′), R^(2′), R^(3′),R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(c) is selected from a group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol that has a terminal group selected from the groupconsisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is—NR^(c)— or —O—; and p is 1 to 3.

In aspects of Formulae (V)-(X),

line represents an indirect bond to a carrier particle (e.g.,nanoparticle) through a suitable spacer, such as, but not limited to PEGspacers. In aspects of Formulae (V)-(X) or (V-B)-(X-B), Payload is aresidue of cytotoxic moiety, and when Payload is a separate molecularentity it contains an amino or hydroxyl group that provides the nitrogenor oxygen atom at Z. In preferred aspects of Formulae (V)-(X) or(V-B)-(X-B), Payload is a residue of cytotoxic moiety.

In aspects of Formulae (V)-(IX) or (V-B)-(IX-B), including thosepreferred aspects described above, R¹ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl,preferably methyl. In preferred aspects of Formulae (V)-(IX) or(V-B)-(IX-B), R¹ is hydrogen. In preferred aspects of Formulae (V)-(IX),R¹ is methyl.

In aspects of Formulae (V), (VII)-(IX), (V-B), (VII-B), or (IX-B),including those preferred aspects described above, R² is hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl. In preferred aspects ofFormulae (V), (VII)-(IX) (V-B), (VII-B), or (IX-B), R² is hydrogen. Inpreferred aspects of Formulae (V), (VII)-(IX). (V-B), or (VII-B)-(IX-B),R² is methyl.

In aspects of Formulae (V), (VI), (VIII), (X), (V-B), (VI-B), (VIII-B)or (X-B), including those preferred aspects described above, R³ and R⁴in in each occurrence is independently hydrogen, halo, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy. Inpreferred aspects of Formulae (V), (VI), (VIII), (X), (V-B), (VI-B),(VIII-B) or (X-B), R³ and R⁴ in each occurrence is independentlyhydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ alkoxy. In preferred aspects of Formulae (V),(VI), (VIII), (X), (V-B), (VI-B), (VIII-B) or (X-B), R³ and R⁴ in eachoccurrence is independently hydrogen, chloro, fluoro, methyl or methoxy.In preferred aspects of Formulae (V), (VI), (VIII), (X), (V-B), (VI-B),(VIII-B) or (X-B), R³ and R⁴ is hydrogen. In preferred aspects ofFormulae (V), (VI), (VIII), (X), (V-B), (VI-B), (VIII-B) or (X-B), R³and R⁴ is fluoro.

In aspects of Formulae (V)-(X) or (V-B)-(X-B), including those preferredaspects described above, R⁵ and R⁶ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl. In preferredaspects of Formulae (V)-(X) or (V-B)-(X-B), R⁵ and R⁶ in each occurrenceis hydrogen. In preferred aspects of Formulae (V)-(X) or (V-B)-(X-B), R⁵and R⁶ in each occurrence is methyl. In certain aspects of Formulae(V)-(X) or (V-B)-(X-B), including those preferred aspects describedabove, R⁷ is selected from the group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl. In preferred aspects ofFormulae (V)-(X) or (V-B)-(X-B), R⁷ is hydrogen. In preferred aspects ofFormulae (V)-(X) or (V-B)-(X-B), R⁷ is methyl.

In certain aspects of Formulae (V), (VI), (V-B), or (VI-B) includingthose preferred aspects described above, R^(a), R^(b) and R^(c) in eachoccurrence is independently hydrogen or substituted or unsubstitutedC₁₋₆ alkyl. In preferred aspects of Formulae (V), (VI), (V-B), or(VI-B), R^(a), R^(b) and R^(c) is hydrogen. In preferred aspects ofFormulae (V), (VI), (V-B), or (VI-B), R^(a), R^(b) and R^(c) is methyl.In preferred aspects of Formulae (V), (VI), (V-B), or (VI-B), R^(a) andR^(b) is hydrogen and R^(c) is methyl. In preferred aspects of Formulae(V), (VI), (V-B), or (VI-B), R^(a) and R^(b) is methyl and R^(c) ishydrogen. In preferred aspects of Formulae (V), (VI), (V-B), or (VI-B),R^(a) is methyl or hydrogen, R^(b) is hydrogen or methyl and R^(c) ishydrogen or methyl.

In aspects of Formulae (VII)-(X) or (VII-B)-(X-B), including thosepreferred aspects described above, R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl. Inpreferred aspects of Formulae (VII)-(X) or (VII-B)-(X-B), R^(c) ishydrogen. In preferred aspects of Formulae (VII)-(X) or (VII-B)-(X-B),R^(c) is methyl.

In aspects of Formula (V) or (V-B), including those preferred aspectsdescribed above, X is absent, —O— or —NR^(a)—. In preferred aspects ofFormula (V) or (V-B), X is absent. In preferred aspects of Formula (V)or (V-B), X is —O—. In preferred aspects of Formula (V) or (V-B), X is—NR^(a)—, wherein R^(a) is selected from a group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl, preferably, R^(a) is methyl.

In aspects of Formulae (V), (VI), (X), (V), (VI) or (X), including thosepreferred aspects described above, X₁ and X₂ are independently —CH— or—N—; X₃ is —CH— and X₄ is —CH—. In preferred aspects of Formulae (V),(VI), (X), (V), (VI) or (X), X₁, X₂, X₃ and X₄ are each —CH—. Inpreferred aspects of Formulae (V), (VI), (X), (V), (VI) or (X), X₁ is—CH—; X₂ is —N—; X₃ is —CH— and X₄ is —CH—. In preferred aspects ofFormulae (V), (VI), (X), (V), (VI) or (X), X₁ is —N—; X₂ is —CH—; X₃ is—CH— and X₄ is —CH—.

In aspects of Formula (V) or (V-B), including those preferred aspectsdescribed above, Y is absent,

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (V) or (V-B), Y isabsent. In preferred aspects of Formula (V) or (V-B), Y is

In preferred aspects of Formula (V) or (V-B), Y is

In preferred aspects of Formula (V) or (V-B), Y is

preferably Y is —CONH₂. In preferred aspects of Formula (V) or (V-B), Yis

In preferred aspects of Formula (VI) or (VI-B), Y is

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (VI) or (VI-B), Y is

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (VI) or (VI-B), Y is

wherein the carbonyl in

is bonded to Z.

In aspects of Formula (VI) or (VI-B), including those preferred aspectsdescribed above, Y₁ is

In certain aspects of Formula (VI) or (VI-B), Y₁ is

preferably, Y₁ is —COOCH₃. In certain aspects of Formula (VI) or (VI-B),Y₁ is

preferably, Y₁ is —CONH₂.

In certain aspects of Formula (VII) or (VII-B), Y is

wherein the carbonyl in or

is bonded to Z.

In certain aspects of Formula (VIII) or (VIII-B), Y is

In certain aspects of Formula (VIII) or (VIII-B), Y is

In aspects of Formulae (V)-(X) or (V-B)-(X-B), including those preferredaspects described above, Z is —NR^(c)— or —O—, preferably, Z is—NR^(c)—, wherein R^(c) is hydrogen or substituted or unsubstituted C₁₋₆alkyl, preferably, R is methyl or preferably, Z is —O—.

In aspects of Formula (V) or (V-B), including those preferred aspectsdescribed above, n is 0 or 1 and q is 1 to 3. In aspects of Formula (V)or (V-B), including those preferred aspects described above, n is 0 andq is 1 to 3. In aspects of Formula (V) or (V-B), including thosepreferred aspects described above, n is 1 and q is 1 to 3.

In aspects of Formulae (V)-(X) or (V-B)-(X-B), including those preferredaspects described above, p is 1 to 3.

In aspects of Formulae (VIII), (IX), (VIII-B), OR (IX-B) including thosepreferred aspects described above, m is 1 to 3.

pH-sensitive Linker-payload Conjugates: In certain aspects of thepresent disclosure, the linker-payload conjugates of Formulae (XI)-(XII)and (XI-B)-(XII-B) relate to pH-sensitive linkers that are conjugated toa cytotoxic agent (drug moiety or payload) such as SN38, analogs ofSN38, exatecan and analogs of exatecan.

A linker-payload conjugate may comprise a compound of Formula (XI)

or a salt thereof, wherein

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z, or Payload is amoiety that is liberated by cleavage of N═C bond, such as

R¹, R² and R³ in each occurrence is independently hydrogen, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R⁴ is substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; and Zis —NR^(c)— or —O—.

A linker-payload conjugate may comprise a compound of Formula (XI-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z or Payload is a moiety that is liberated by cleavage of N═Cbond, such as

R¹, R² and R³ in each occurrence is independently hydrogen, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R⁴ is substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ cycloalkyl; R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R^(c) is selected from a group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

and Z is —NR— or —O—.

A linker-payload conjugate may comprise a compound of Formula (XII)

or a salt thereof, wherein

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group; Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z, or Payload is amoiety that is liberated by cleavage of N═C bond, such as

R¹, R² and R³ in each occurrence is independently hydrogen, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R⁴ is substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; Z is—NR^(c)— or —O—; and n₁ is 0 to 3.

A linker-payload conjugate may comprise a compound of Formula (XII-B)

or a salt thereof, wherein: Payload is a residue of a drug moiety or apro-drug moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z or Payload is a moiety that is liberated by cleavage of N═Cbond, such as

R¹, R² and R³ in each occurrence is independently hydrogen, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R⁴ is substituted or unsubstituted C₁₋₆ alkyl or substitutedor unsubstituted C₁₋₆ cycloalkyl; R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl; R^(c) is selected from a group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

Z is —NR^(c)— or —O—; and n₁ is 0 to 3.

In aspects of Formulae (XI)-(XII), line represents an indirect bond to acarrier particle (e.g., nanoparticle) through a suitable spacer, suchas, but not limited to PEG spacers. In aspects of Formulae (XI), (XII),(XI-B), or (XII-B), Payload is a residue of cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z. In preferredaspects of Formulae (XI), (XII), (XI-B), or (XII-B), Payload is aresidue of cytotoxic moiety.

In other preferred aspects of Formula (XI) or (XI-B), Payload is amoiety that is liberated by cleavage of N═C bond, such as

In other preferred aspects of Formula (XII) or (XII-B), Payload is amoiety that is liberated by cleavage of N═C bond, such as

In aspects of Formulae (XI), (XII), (XI-B), or (XII-B), including thosepreferred aspects described above, R¹, R² and R³ in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl and R⁴ is substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆cycloalkyl. In preferred aspects of Formulae (XI), (XII), (XI-B), or(XII-B), including those preferred aspects described above, R¹ and R² ineach occurrence is hydrogen, R³ is hydrogen, R⁴ is methyl and R^(c) ishydrogen or substituted or unsubstituted C₁₋₆ alkyl, preferably R^(c) ishydrogen or methyl. In preferred aspects of Formulae (XI), (XII),(XI-B), or (XII-B), including those preferred aspects described above,R¹ and R² in each occurrence is methyl, R³ is hydrogen, R⁴ is methyl andR^(c) is hydrogen or substituted or unsubstituted C₁₋₆ alkyl, preferablyR^(c) is hydrogen or methyl. In aspects of Formulae (XI), (XII), (XI-B),or (XII-B), including those preferred aspects described above, Z is—NR^(c)— or —O—, preferably, Z is —NR—, wherein R^(c) is hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl, preferably, R^(c) is methyl orpreferably, Z is —O—.

In aspects of Formula (XII) or (XII-B), including those preferredaspects described above, n1 is 0 to 3. In certain aspects of Formula(XII) or (XII-B), including those preferred aspects described above, n1is 0. In certain aspects of Formula (XII) or (XII-B), including thosepreferred aspects described above, n1 is 1. In certain aspects ofFormula (XII) or (XII-B), including those preferred aspects describedabove, n1 is 2. In certain aspects of Formula (XII) or (XII-B),including those preferred aspects described above, n1 is 3.

Payload: Payloads suitable for conjugation to a carrier particle (e.g.,nanoparticle) may comprise a functional group that can facilitateconjugation to the carrier particle, e.g., via a linker and/or spacergroup. For example, the linker-payload conjugates of Formulae (I)-(XII)or (I-B)-(XII-B), the payload may have a chemically reactive functionalgroup that is bonded to the linker, wherein the functional group isselected from the group consisting of an amine (e.g., primary amine,secondary amine), hydroxyl, sulfhydryl, and carboxyl.

In Formulae (I)-(XII) or (I-B)-(XII-B), the payload has either an amineor a hydroxyl functional group for conjugation to the linker moiety. Inpreferred aspects, Payload is a residue of a cytotoxic moiety, and whenPayload is a separate molecular entity it contains an amino or hydroxylgroup that provides the nitrogen or oxygen atom at Z. In preferredaspects of the present disclosure, Payload is a cytotoxic agent selectedfrom a group consisting of dihydrofolate reductase inhibitors,thymidylate synthase inhibitors, and topoisomerase inhibitors. In someaspects, the payload is a topoisomerase inhibitor. For example, inFormulae (I)-(XII) or (I-B)-(XII-B), Payload can be a topoisomeraseinhibitor selected from a group consisting of SN38, analogs of SN38,exatecan and analogs of exatecan. In Formulae (I)-(XII) or(I-B)-(XII-B), the payload can be SN38. In Formulae (I)-(XII) or(I-B)-(XII-B), the payload can be an analog of SN38, such as thosedescribed in the present disclosure. In Formulae (I)-(XII) or(I-B)-(XII-B), the payload can be exatecan. In Formulae (I)-(XII) or(I-B)-(XII-B), the payload can be an analog of exatecan, such as thosedescribed in the present disclosure.

In certain aspects of Formulae (I)-(IV) or (I-B)-(IV-B), when A is adipeptide, Payload is selected from a group consisting of SN38, analogsof SN38, exatecan and analogs of exatecan. For example, in Formulae(I)-(IV) or (I-B)-(IV-B), when A is a dipeptide, Payload may be SN38. Insome aspects of Formulae (I)-(IV) or (I-B)-(IV-B), when A is adipeptide, Payload is an analog of SN38, such as those described in thepresent disclosure. In other aspects of Formulae (I)-(IV) or(I-B)-(IV-B), when A is a dipeptide, Payload is Exatecan. In otheraspects of Formulae (I)-(IV) or (I-B)-(IV-B), when A is a dipeptide,Payload is an analog of Exatecan, such as those described in the presentdisclosure.

In Formulae (I)-(IV) or (I-B)-(IV-B), Payload may comprise an analog ofSN38 of formula (105)

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (I)-(IV) or (I-B)-(IV-B), andone or more hydrogen atoms may be absent, valency permitting).

In Formulae (V)-(X) or (V-B)-(X-B), Payload may comprise an analog ofSN38 selected from a group consisting of

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (V)-(X) or (V-B)-(X-B), and oneor more hydrogen atoms may be absent, valency permitting).

In Formulae (XI)-(XII) or (XI-B)-(XII-B), the Payload can be an analogof SN38 selected from a group consisting of

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (XI)-(XII) or (XI-B)-(XII-B),and one or more hydrogen atoms may be absent, valency permitting).

In Formulae (I)-(IV) or (I-B)-(IV-B), the Payload can be an analog ofExatecan of formula:

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (I)-(IV) or (I-B)-(IV-B), andone or more hydrogen atoms may be absent, valency permitting).

In Formulae (V)-(X) or (V-B)-(X-B), the Payload can be an analog of SN38selected from a group consisting of

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (V)-(X) or (V-B)-(X-B), and oneor more hydrogen atoms may be absent, valency permitting).

In Formulae (XI)-(XII) or (XI-B)-(XII-B), the Payload can be an analogof exatecan of formula

It will be understood that a functional group (e.g., an —OH or —NH₂group) may be used to conjugate the payload to the linker, e.g., theoxygen or nitrogen may be Z in Formulae (XI)-(XII) or (XI-B)-(XII-B),and one or more hydrogen atoms may be absent, valency permitting).

Exemplary Linker-Payload Conjugates: Representative linker-payloadconjugates of the present disclosure include, but are not limited to thefollowing sub-structures, wherein

line represents a direct bond to a carrier particle (e.g., nanoparticle)or an indirect bond to the carrier particle (e.g., nanoparticle) througha spacer group. Suitable spacer groups include, but are not limited to aPEG spacer, or an alkylene spacer (e.g., methylene spacer), which mayfurther comprise heteroatoms, or cyclic groups (e.g., heterocyclylenegroups). In preferred aspects, the spacer group is a PEG spacer.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (I) or (I-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (IV) or (IV-1B) of thepresent disclosure may include the following sub-structure.

An exemplary linker-payload conjugate of Formula (V) or (V-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (VII) or (VII-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (XI) or (XI-B) of thepresent disclosure may include one of the following sub-structures.

An exemplary linker-payload conjugate of Formula (XII) or (XII-B) of thepresent disclosure may include one of the following sub-structures.

The protease-cleavable linker-payload conjugates of the presentdisclosure can also include, but are not limited to including, one ofthe following sub-structures, that can be prepared using the schemes andmethods disclosed herein.

Other linker-payload conjugates of the present disclosure may includeone of the following sub-structures.

Yet another linker-payload conjugates of the present disclosure mayinclude one of the following sub-structures.

Other linker-payload conjugates of the present disclosure may includeone of the following sub-structures.

Linkers: The linkers of this disclosure (or precursors thereof) cancontain reactive groups at both ends of the molecule. The reactivegroups can be selected to allow conjugation to any payload (e.g.,cytotoxic payload) at one end and also facilitate conjugation to anydrug-delivery system (e.g., nanoparticle) at the other end, e.g., via aspacer group. This disclosure relates to linkers of Formulae(I-A)-(X-A).

In Formulae (I-A)-(X-A), there are functional groups at each end of themolecule. The functional groups are Z₁ and Z₂. Z₁ can be any functionalgroup on one end of the linker moiety that connects to any desirablepayload. Z₂ can be any functional group on other end of the linkermoiety that allows conjugation to a spacer group, such as afunctionalized polyethylene glycol or a C₅-C₆ alkyl chain. For example,the linker can connect to any desirable payload via a chemicallyreactive functional group that is a part of the payload such as aprimary or secondary amine, hydroxyl, sulfhydryl, or carboxyl group. Forexample, the linker can be conjugated to a functionalized polyethyleneglycol or a C₅-C₆ alkyl chain via a chemically reactive functional groupthat is a part of the linker such as a primary or secondary amine orcarboxyl group.

Protease-cleavable Linkers: Proteases are involved in all stages ofcancer disease from tumor cells growth and survival, to angiogenesis andinvasions. Therefore, they can be utilized to treat cancer as selectivetriggers towards activation of linker/payload system. This disclosurerelates to linkers that are cleavable by the action of proteases therebyreleasing the free payload. Lysosomal proteases such as cathepsin B andserine proteases such as cathepsin A or tripeptidyl-peptidase I havebeen extensively studied in the context of prodrug development.Proteolytic enzymes such as caspases are also well-known to be utilizedas biological triggers for the selective activation of payload or forspecific cargo delivery to a target cell such as a cancer cell.

In certain aspects of the present disclosure, the linkers of Formulae(I-A)-(IV-A) relate to protease-cleavable linkers.

A linker can comprise a compound of Formula (I-A)

wherein, A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala; or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar (SEQID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; R¹ and R² ineach occurrence is independently hydrogen, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy, or hydroxy; R³and R⁴ in each occurrence is independently hydrogen, halo, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy;R⁵ is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted C₅-6 heterocycloalkyl, withthe proviso that, when A is a dipeptide, R⁵ is H; R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl, or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) in each occurrence isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ isa functionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

X is absent, —O—, —CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z₁, with the proviso that, when Y is

X is absent and n is 1, with the proviso that, when Y is

X is absent and n is 0, with the proviso that, when Y is

X is absent and n is 0 or 1, with the proviso that, when X is —CO—, Y isabsent and n is 0; X₃ is —CH—; X₄ is —CH—; Z₁ is a functional groupselected from the group consisting of halo, hydroxy, —OSO₂—CH₃,—OSO₂CF₃, 4-nitrophenoxy, —COCl, and —COOH; Z₂ is a functional groupselected from the group consisting of —NH₂, —NHR^(c), and —COOH; or Z₂is —C(O)-T₁; n is 0 or 1; and q is 1 to 3.

A linker may comprise a compound of Formula (II-A)

wherein, A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar (SEQID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; R¹ isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³and R⁴ in each occurrence is independently hydrogen, halo, substitutedor unsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy;R⁵ is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl; and substituted or unsubstituted C₅₋₆ heterocycloalkyl, withthe proviso that, when A is a dipeptide, R⁵ is H; R^(1′), R^(2′), R²′,R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) in each occurrence isindependently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ isa functionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is

Z₁ is a functional group selected from the group consisting of halo,mesylate, and tosylate; and Z₂ is a functional group selected from thegroup consisting of —NH₂, —NHR^(c), and —COOH; or Z₂ is —C(O)-T₁.

A linker may comprise a compound of Formula (III-A)

wherein, A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala; or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar (SEQID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; R³ and R⁴ ineach occurrence is independently hydrogen, halo, substituted orunsubstituted C₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted C₅₋₆ heterocycloalkyl, withthe proviso that, when A is a dipeptide, R⁵ is H; R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl; R^(c) is selected from the group consisting of hydrogenor substituted or unsubstituted C₁₋₆ alkyl; T₁ is a functionalizedpolyethylene glycol or a C₅-C₆ alkyl chain that has a terminal groupselected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z₁ is afunctional group selected from the group consisting of —N═C═O, —O—COCl,—O—COO—4-nitrophenyl, —NH—COO—4-nitrophenyl, and —CHO; Z₂ is afunctional group selected from the group consisting of —NH₂, —NHR^(c),and —COOH or Z₂ is —C(O)-T₁.

A linker may comprise a compound of Formula (IV-A)

Z ₂-A-Z ₁  (IV-A)

wherein, A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala; or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar (SEQID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Phe-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Ala-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, and Trp-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid; R^(1′),R^(2′) R^(3′), R⁴′ and R^(5′) in each occurrence is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl or substituted orunsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from the groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁ isa functionalized polyethylene glycol or a C₅-C₆ alkyl chain that has aterminal group selected from the group consisting of azide,

Z₁ is —COOH; and Z₂ is a functional group selected from the groupconsisting of —NH₂, —NHR^(c), and —COOH or Z₂ is —C(O)-T₁.

In certain aspects of Formulae (I-A)-(IV-A), A is a dipeptide or atetrapeptide. In certain aspects of (I-A)-(IV-A), A is a dipeptideselected from the group consisting of Val-Cit, Phe-Lys, Trp- Lys,Asp-Lys, Val- Lys, Val-Arg, and Val-Ala. In preferred aspects ofFormulae (I-A)-(IV-A), A is Val-Cit. In preferred aspects of Formulae(I-A)-(IV-A), A is Val-Lys. In preferred aspects of Formulae(I-A)-(IV-A), A is Phe-Lys.

In certain aspects of Formulae (I-A)-(IV-A), including those preferredaspects described above, A is a tetrapeptide selected from the groupconsisting of Val-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar,Val-Lys-Gly-Sar (SEQ ID NO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11),Val-Phe-Gly-Pro (SEQ ID NO: 12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQID NO: 14), Val-Ala-Gly-Pro (SEQ ID NO: 15), Val-Cit-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Val-Phe-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Ala-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Phe-Lys-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, and Trp-Lys-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid. In preferred aspects of Formulae (I-A)-(IV-A), A is atetrapeptide selected from a group consisting of Val-Phe-Gly-Sar (SEQ IDNO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10),Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12),Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), and Val-Ala-Gly-Pro(SEQ ID NO: 15). In preferred aspects of Formulae (I-A)-(IV-A), A isVal-Lys-Gly-Sar (SEQ ID NO: 10). In preferred aspects of Formulae(I-A)-(IV-A), A is Val-Ala-Gly-Sar (SEQ ID NO: 11). In preferred aspectsof Formulae (I-A)-(IV-A), A is Val-Phe-Gly-Pro (SEQ ID NO: 12). Inpreferred aspects of Formulae (I-A)-(IV-A), A is Val-Cit-Gly-Pro. Inpreferred aspects of Formulae (I-A)-(IV-A), A is Val-Lys-Gly-Pro (SEQ IDNO: 14). In preferred aspects of Formulae (I-A)-(IV-A), A isVal-Ala-Gly-Pro (SEQ ID NO: 15).

In aspects of Formula (I-A), including those preferred aspects describedabove, R¹ and R² in each occurrence is independently hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy, or hydroxy. In preferred aspects of Formula (I-A), R¹ andR² is hydrogen. In preferred aspects of Formula (I-A), R¹ and R² ismethyl. In preferred aspects of Formula (I-A), R¹ is hydrogen and R² ishydroxy. In preferred aspects of Formula (I-A), R¹ is hydrogen and R² ismethyl. In preferred aspects of Formula (I-A), R¹ is hydroxy and R² ismethyl.

In aspects of Formula (II-A), including those preferred aspectsdescribed above, R¹ is selected from a group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl. In preferred aspects of Formula(II-A), R¹ is hydrogen. In preferred aspects of Formula (II-A), R¹ ismethyl.

In aspects of Formulae (I-A), (II-A) and (III-A), including thosepreferred aspects described above, R³ and R⁴ in each occurrence isindependently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ alkoxy. In preferred aspects ofFormulae (I-A), (II-A) and (III-A), R³ and R⁴ in each occurrence isindependently hydrogen, chloro, fluoro, methyl or methoxy. In preferredaspects of Formulae (I-A), (II-A) and (III-A), R³ and R⁴ is hydrogen. Inpreferred aspects of Formulae (I-A), (II-A) and (III-A), R³ and R⁴ isfluoro.

In aspects of Formulae (I-A), (II-A) and (III-A), including thosepreferred aspects described above, R⁵ is selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁₋₆ alkyl;substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅-6 heterocycloalkyl, with the provisothat, when A is a dipeptide, R⁵ is H. In certain aspects of Formulae(I-A), (II-A) and (III-A), including those preferred aspects describedabove, R⁵ is selected from the group consisting of hydrogen, methyl,cyclopropyl, phenyl or a substituted phenyl. In preferred aspects ofFormulae (I-A), (II-A) and (III-A), R⁵ is hydrogen. In preferred aspectsof Formulae (I-A), (II-A) and (III-A), when A is a dipeptide, R⁵ is H.

In certain aspects of Formulae (I-A) and (II-A), including thosepreferred aspects described above, R^(a), R^(b) and R^(c) in eachoccurrence is independently hydrogen or substituted or unsubstitutedC₁₋₆ alkyl. In preferred aspects of Formulae (I-A) and (II-A), R^(a),R^(b) and R^(c) is hydrogen. In preferred aspects of Formulae (I-A) and(II-A), R^(a), R^(b) and R^(c) is methyl. In preferred aspects ofFormula (II-A), R^(b) is hydrogen and R^(c) is methyl. In aspects ofFormulae (III-A)-(IV-A), including those preferred aspects describedabove, R^(c) is selected from a group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl. In aspects of Formulae(III-A)-(IV-A), R^(c) is hydrogen. In aspects of Formulae(III-A)-(IV-A), R^(c) is methyl.

In aspects of Formula (I-A), including those preferred aspects describedabove, X is absent, —O—, —CO— or —NR^(a)—. In preferred aspects ofFormula (I-A), X is absent. In preferred aspects of Formula (I-A), X is—O— and n is 0. In preferred aspects of Formula (I-A), X is —CO— and nis 0. In preferred aspects of Formula (I-A), X is —NR^(a)—, whereinR^(a) is selected from a group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl, preferably, R^(a) is methyl.

In preferred aspects of Formula (I-A), Y is absent,

wherein the carbonyl in

is bonded to Z; with the proviso that, when Y is

X is absent and n is 1; with the proviso that, when Y is

X is absent and n is 0; with the proviso that, when Y is

X is absent and n is 0 or 1 and with the proviso that, when X is —CO—, Yis absent and n is 0. In preferred aspects of Formula (I-A), Y isabsent. In preferred aspects of Formula (I-A), Y is

In preferred aspects of Formula (I-A), Y is

In preferred aspects of Formula (I-A), Y is

preferably Y is —CONH₂. In preferred aspects of Formula (I-A), Y is

In preferred aspects of Formula (I-A), Y is

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (I-A), Y is

wherein the carbonyl in

is bonded to Z. In preferred aspects of Formula (I-A), Y is

wherein the carbonyl in

is bonded to Z. In aspects of Formula (II-A), including those preferredaspects described above Y₁ is

In certain aspects of Formula (II-A), Y₁ is

preferably, Y₁ is —COOCH₃. In certain aspects of Formula (II-A), Y₁ is

preferably, Y₁ is —CONH₂.

In aspects of Formulae (I-A), (II-A) and (III-A), including thosepreferred aspects described above, X₁ and X₂ are independently —CH— or—N—; X₃ is —CH— and X₄ is —CH—. In preferred aspects of Formulae (I-A),(II-A) and (III-A), X₁, X₂, X₃ and X₄ are each —CH—. In preferredaspects of Formulae (I-A), (II-A) and (III-A), X₁ is —CH—; X₂ is —N—; X₃is —CH— and X₄ is —CH—. In preferred aspects of Formulae (I-A), (II-A)and (III-A), X₁ is —N—; X₂ is —CH—; X₃ is —CH— and X₄ is —CH—.

In aspects of Formula (I-A), including those preferred aspects describedabove, Z₁ is a functional group selected from the group consisting ofhalo, hydroxy, —OSO₂—CH₃, —OSO₂CF₃, 4-nitrophenoxy, —COCl, and —COOH. Inpreferred aspects of Formula (I-A), Z₁ is halo, preferably chloro orfluoro. In preferred aspects of Formula (I-A), Z₁ is hydroxy. Inpreferred aspects of Formula (I-A), Z₁ is 4-nitrophenoxy. In preferredaspects of Formula (I-A), Z₁ is —COCl. In preferred aspects of Formula(I-A), Z₁ is —COOH. In aspects of Formula (II-A), including thosepreferred aspects described above, Z₁ is a functional group selectedfrom the group consisting of halo, mesylate, and tosylate. In preferredaspects of Formula (II-A), Z₁ is halo, preferably chloro or bromo. Inpreferred aspects of Formula (II-A), Z₁ is mesylate. In preferredaspects of Formula (II-A), Z₁ is tosylate. In aspects of Formula(III-A), including those preferred aspects described above, Z₁ is afunctional group selected from the group consisting of —N═C═O, —O—COCl,—O—COO—4-nitrophenyl, —NH—COO—4-nitrophenyl, and —CHO. In preferredaspects of Formula (III-A), Z₁ is —N═C═O. In preferred aspects ofFormula (III-A), Z₁ is —O—CO—C₁. In preferred aspects of Formula(III-A), Z₁ is —O—COO—4-nitrophenyl. In preferred aspects of Formula(III-A), Z₁ is —NH—COO—4-nitrophenyl. In preferred aspects of Formula(III-A), Z₁ is —CHO. In aspects of Formula (IV-A), including thosepreferred aspects described above, Z₁ is —COOH.

In aspects of Formulae (I-A)-(IV-A), including those preferred aspectsdescribed above, Z₂ is a functional group selected from the groupconsisting of —NH₂, —NHR^(c), and —COOH or Z₂ is —C(O)-T₁, wherein T₁ isas defined in Formulae (I-A)-(IV-A). In preferred aspects of Formulae(I-A)-(IV-A), Z₂ is —NH₂, —NHR¹, and —COOH. In preferred aspects ofFormulae (I-A)-(IV-A), Z₂ is —NHR^(c), wherein R^(c) is preferablymethyl. In preferred aspects of Formulae (I-A)-(IV-A), Z₂ is —COOH. Inaspects of Formulae (I-A)-(IV-A), including those preferred aspectsdescribed above, Z₂ is —C(O)-T₁, wherein T₁ is as defined in Formulae(I-A)-(IV-A).

In aspects of Formula (I-A), including those preferred aspects describedabove, n is 0 or 1 and q is 1 to 3. In aspects of Formula (I-A),including those preferred aspects described above, n is 0 and q is 1 to3. In aspects of Formula (I-A), including those preferred aspectsdescribed above, n is 1 and q is 1 to 3.

Redox-sensitive Linkers: This disclosure relates to redox-sensitivelinkers that may also be referred to as redox-responsive linkers. Theredox-sensitive linkers may include a disulfide bond that can be reduced(e.g., by reducing glutathione (GSH)) into sulfhydryl groups. This inturn causes the degradation of the linker and facilitates the release ofpayload or an active analog of a payload. In certain aspects of thepresent disclosure, the linkers of Formulae (V-A)-(X-A) relate toredox-sensitive linkers.

A linker may comprise a compound of Formula (V-A)

wherein, R¹, R², R⁵ and R⁶ in each occurrence is independently hydrogenor substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in each occurrenceis independently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkylor substituted or unsubstituted C₁₋₆ alkoxy; R⁷ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;R^(1′), R²′, R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) ineach occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; T₁ is a functionalized polyethylene glycol ora C₅-C₆ alkyl chain that has a terminal group selected from the groupconsisting of azide,

X is absent, —O— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z₁; X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄is —CH—; Z₁ is a functional group selected from the group consisting ofhalo, hydroxy, 4-nitrophenoxy, —O—SO₂—CH₃ and —O—SO₂—CF₃; Z₂ is afunctional group selected from the group consisting of —NH₂, —NHR^(c),and —COOH, or Z₂ is —NR⁷—C(O)-T₁; n is 0 or 1; p is 1 to 3; and q is 1to 3.

A linker can comprise a compound of Formula (VI-A)

wherein, R₁, R⁵ and R⁶ in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in each occurrence isindependently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ alkoxy; R⁷ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl; R¹′,R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl or substituted orunsubstituted C₁₋₆ cycloalkyl; R^(a), R^(b) and R^(c) in each occurrenceis independently hydrogen or substituted or unsubstituted C₁₋₆ alkyl; T₁is a functionalized polyethylene glycol or a C₅-C₆ alkyl chain that hasa terminal group selected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Y₁ is

Z₁ is a functional group selected from the group consisting of halo,mesylate, triflate, and tosylate; Z₂ is a functional group selected fromthe group consisting of —NH₂, —NHR^(c), and —COOH, or Z₂ is—NR⁷—C(O)-T₁; and p is 1 to 3.

A linker may comprise a compound of Formula (VII-A)

wherein, R₁, R², R⁵ and R⁶ in each occurrence is independently hydrogenor substituted or unsubstituted C₁₋₆ alkyl; R⁷ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from thegroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;T₁ is a functionalized polyethylene glycol or a C₅-C₆ alkyl chain thathas a terminal group selected from the group consisting of azide,

Y is

wherein the carbonyl in or

is bonded to Z₁; Z₁ is a functional group selected from the groupconsisting of halo, hydroxy, 4-nitrophenoxy, —O—SO₂—CH₃, and —O—SO₂—CF₃;Z₂ is a functional group selected from the group consisting of —NH₂,—NHR⁷, and —COOH, or Z₂ is —NR⁷—C(O)-T₁; and p is 1 to 3.

A linker may comprise a compound of Formula (VIII-A)

wherein, R¹, R², R³, R⁵ and R⁶ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R⁴ is halo; R⁷ isselected from a group consisting of hydrogen or substituted orunsubstituted C₁₋₆ alkyl; R^(1′), R²′, R^(3′), R^(4′) and R^(5′) in eachoccurrence is independently hydrogen, substituted or unsubstituted C₁₋₆alkyl or substituted or unsubstituted C₁₋₆ cycloalkyl; R^(c) is selectedfrom the group consisting of hydrogen or substituted or unsubstitutedC₁₋₆ alkyl; T₁ is a functionalized polyethylene glycol or a C₅-C₆ alkylchain that has a terminal group selected from the group consisting ofazide,

Z₁ is halo; Z₂ is a functional group selected from the group consistingof —NH₂, —NHR^(c), and —COOH, or Z₂ is —NR⁷—C(O)-T₁; m is 1 to 3; and pis 1 to 3.

A linker may comprise a compound of Formula (IX-A)

wherein, R¹, R², R⁵ and R⁶ in each occurrence is independently hydrogenor substituted or unsubstituted C₁₋₆ alkyl; R⁷ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from thegroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;T₁ is a functionalized polyethylene glycol or a C₅-C₆ alkyl chain thathas a terminal group selected from the group consisting of azide,

Z₁ is a functional group selected from the group consisting of —COOH,—O—COCl and —O—COO—4-nitrophenoxy; Z₂ is a functional group selectedfrom the group consisting of —NH₂, —NHR^(c), and —COOH, or Z₂ is—NR⁷—C(O)-T₁; m is 1 to 3; and p is 1 to 3.

A linker may comprise a compound of Formula (X-A)

wherein, R³ and R⁴ in each occurrence is independently hydrogen, halo,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ alkoxy; R⁵ and R⁶ in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; R⁷ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl; R^(c) is selected from thegroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;T₁ is a functionalized polyethylene glycol or a C₅-C₆ alkyl chain thathas a terminal group selected from the group consisting of azide,

X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z₁ is afunctional group selected from the group consisting of —N═C═O, —CHO,—COOH, and —COCl; Z₂ is a functional group selected from the groupconsisting of —NH₂, —NHR^(c), and —COOH, or Z₂ is —NR⁷—C(O)-T₁; and p is1 to 3.

In aspects of Formulae (V-A)-(IX-A), R¹ is selected from a groupconsisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl. Inpreferred aspects of Formulae (V-A)-(IX-A), R¹ is hydrogen. In preferredaspects of Formulae (V-A)-(IX-A), R¹ is methyl. In aspects of Formulae(V-A), (VII-A)-(IX-A), including those preferred aspects describedabove, R² is hydrogen or substituted or unsubstituted C₁₋₆ alkyl. Inpreferred aspects of Formulae (V-A), (VII-A)-(IX-A), R² is hydrogen. Inpreferred aspects of Formulae (V-A), (VII-A)-(IX-A), R² is methyl. Inaspects of Formulae (V-A), (VI-A), (VIII-A) and (X-A), including thosepreferred aspects described above, R³ and R⁴ in in each occurrence isindependently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ alkoxy. In preferred aspects ofFormulae (V-A), (VI-A), (VIII-A) and (X-A), R³ and R⁴ in each occurrenceis independently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkylor substituted or unsubstituted C₁₋₆ alkoxy. In preferred aspects ofFormulae (V-A), (VI-A), (VIII-A) and (X-A), R³ and R⁴ in each occurrenceis independently hydrogen, chloro, fluoro, methyl or methoxy. Inpreferred aspects of Formulae (V-A), (VI-A), (VIII-A) and (X-A), R³ andR⁴ is hydrogen. In preferred aspects of Formulae (V-A), (VI-A), (VIII-A)and (X-A), R³ and R⁴ is fluoro. In aspects of Formulae (V-A)-(X-A),including those preferred aspects described above, R⁵ and R⁶ in eachoccurrence is independently hydrogen or substituted or unsubstitutedC₁₋₆ alkyl. In preferred aspects of Formulae (V-A)-(X-A), R⁵ and R⁶ ineach occurrence is hydrogen. In preferred aspects of Formulae(V-A)-(X-A), R⁵ and R⁶ in each occurrence is methyl. In certain aspectsof Formulae (V-A)-(X-A), including those preferred aspects describedabove, R⁷ is selected from the group consisting of hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl. In preferred aspects ofFormulae (V-A)-(X-A), R⁷ is hydrogen. In preferred aspects of Formulae(V-A)-(X-A), R⁷ is methyl.

In certain aspects of Formulae (V-A) and (VI-A), including thosepreferred aspects described above, R^(a), R^(b) and R^(c) in eachoccurrence is independently hydrogen or substituted or unsubstitutedC₁₋₆ alkyl. In preferred aspects of Formulae (V-A) and (VI-A), R^(a),R^(b) and R^(c) is hydrogen. In preferred aspects of Formulae (V-A) and(VI-A), R^(a), R^(b) and R^(c) is methyl. In preferred aspects ofFormulae (V-A) and (VI-A), R^(a) and R^(b) is hydrogen and R^(c) ismethyl. In preferred aspects of Formulae (V-A) and (VI-A), R^(a) andR^(b) is methyl and R^(c) is hydrogen. In preferred aspects of Formulae(V-A) and (VI-A), R^(a) is methyl or hydrogen, R^(b) is hydrogen ormethyl and R^(c) is hydrogen or methyl. In aspects of Formulae(VII-A)-(X-A), including those preferred aspects described above, R^(c)is selected from a group consisting of hydrogen or substituted orunsubstituted C₁₋₆ alkyl. In preferred aspects of Formulae(VII-A)-(X-A), R^(c) is hydrogen. In preferred aspects of Formulae(VII-A)-(X-A), R^(c) is methyl.

In aspects of Formula (V-A), including those preferred aspects describedabove, X is absent, —O— or —NR^(a)—. In preferred aspects of Formula(V-A), X is absent. In preferred aspects of Formula (V-A), X is —O—. Inpreferred aspects of Formula (V-A), X is —NR^(a)—, wherein R^(a) isselected from a group consisting of hydrogen or substituted orunsubstituted C₁₋₆ alkyl, preferably, R^(a) is methyl.

In aspects of Formulae (V-A), (VI-A) and (X-A), including thosepreferred aspects described above, X₁ and X₂ are independently —CH— or—N—; X₃ is —CH— and X₄ is —CH—. In preferred aspects of Formulae (V-A),(VI-A) and (X-A), X₁, X₂, X₃ and X₄ are each —CH—. In preferred aspectsof Formulae (V-A), (VI-A) and (X-A), X₁ is —CH—; X₂ is —N—; X₃ is —CH—and X₄ is —CH—. In preferred aspects of Formulae (V-A), (VI-A) and(X-A), X₁ is —N—; X₂ is —CH—; X₃ is —CH— and X₄ is —CH—.

In aspects of Formula (V-A), including those preferred aspects describedabove, Y is absent,

wherein the carbonyl in

is bonded to Z₁. In preferred aspects of Formula (V-A), Y is absent. Inpreferred aspects of Formula (V-A), Y is

In preferred aspects of Formula (V-A), Y is

In preferred aspects of Formula (V-A), Y is

preferably Y is —CONH₂. In preferred aspects of Formula (V-A), Y is

In preferred aspects of Formula (V-A), Y is

wherein the carbonyl in

is bonded to Z₁. In preferred aspects of Formula (V-A), Y is

wherein the carbonyl in

is bonded to Z₁. In preferred aspects of Formula (VI-A), Y is

wherein the carbonyl in

is bonded to Z₁. In preferred of Formula (VI-A), including thosepreferred aspects described above, Y₁ is

In preferred aspects of Formula (VI-A), Y₁ is

preferably, Y₁ is —COOCH₃. In certain aspects of Formula (VI-A), Y₁ is

preferably, Y₁ is —CONH₂. In certain aspects of Formula (VII-A), Y is

In certain aspects of Formula (VII-A), Y is

In certain aspects of Formula (VII-A), Y is

In aspects of Formulae (V-A) and (VII-A), including those preferredaspects described above, Z₁ is a functional group selected from thegroup consisting of halo, hydroxy, 4-nitrophenoxy, —O—SO₂—CH₃ and—O—SO₂—CF₃. In preferred aspects of Formula (V-A), Z₁ is halo,preferably chloro or fluoro. In preferred aspects of Formula (V-A), Z₁is hydroxy. In preferred aspects of Formula (V-A), Z₁ is 4-nitrophenoxy.In preferred aspects of Formula (V-A), Z₁ is —O—SO₂—CH₃. In preferredaspects of Formula (V-A), Z₁ is —O—SO₂—CF₃.

In preferred aspects of Formula (VII-A), Z₁ is halo, preferably chloroor fluoro. In preferred aspects of Formula (VII-A), Z₁ is hydroxy. Inpreferred aspects of Formula (VII-A), Z₁ is 4-nitrophenoxy. In preferredaspects of Formula (VII-A), Z₁ is —O—SO₂—CH₃. In preferred aspects ofFormula (VII-A), Z₁ is —O—SO₂—CF₃. In aspects of Formula (VI-A),including those preferred aspects described above, Z₁ is a functionalgroup selected from the group consisting of halo, mesylate, triflate,and tosylate. In preferred aspects of Formula (VI-A), Z₁ is halo,preferably chloro or fluoro. In preferred aspects of Formula (VI-A), Z₁is mesylate. In preferred aspects of Formula (VI-A), Z₁ is triflate. Inpreferred aspects of Formula (VI-A), Z₁ is tosylate. In aspects ofFormula (VIII-A), including those preferred aspects described above, Z₁is halo, preferably chloro or fluoro. In aspects of Formula (IX-A),including those preferred aspects described above, Z₁ is a functionalgroup selected from the group consisting of —COOH, —O—COCl and—O—COO—4-nitrophenoxy. In preferred aspects of Formula (IX-A), Z₁ is—COOH. In preferred aspects of Formula (IX-A), Z₁ is —O—CO—C₁. Inpreferred aspects of Formula (IX-A), Z₁ is —O—COO—4-nitrophenyl. Inaspects of Formula (X-A), including those preferred aspects describedabove, Z₁ is a functional group selected from the group consisting of—N═C═O, —CHO, —COOH, and —COCl. In preferred aspects of Formula (X-A),Z₁ is —N═C═O. In preferred aspects of Formula (X-A), Z₁ is —CHO. Inaspects of Formula (X-A), Z₁ is —COOH. In preferred aspects of Formula(X-A), Z₁ is —COCl.

In aspects of Formulae (V-A)-(X-A), including those preferred aspectsdescribed above, Z₂ is a functional group selected from the groupconsisting of —NH₂, —NHR¹, and —COOH, or Z₂ is —NR⁷—C(O)-T₁, wherein R⁷and T₁ are as defined in Formulae (V-A)-(X-A). In preferred aspects ofFormulae (V-A)-(X-A), Z₂ is —NH₂, —NHR^(c), and —COOH. In preferredaspects of Formulae (V-A)-(X-A), Z₂ is —NHR^(c), wherein R^(c) ispreferably methyl. In preferred aspects of Formulae (V-A)-(X-A), Z₂ is—COOH. In preferred aspects of Formulae (V-A)-(X-A), Z₂ is —NR⁷—C(O)-T₁,wherein R⁷ and T₁ are as defined in Formulae (V-A)-(X-A).

In aspects of Formula (V-A), including those preferred aspects describedabove, n is 0 or 1 and q is 1 to 3. In aspects of Formula (V-A),including those preferred aspects described above, n is 0 and q is 1 to3. In aspects of Formula (V-A), including those preferred aspectsdescribed above, n is 1 and q is 1 to 3. In aspects of Formulae(V-A)-(X-A), including those preferred aspects described above, p is 1to 3. In aspects of Formulae (VIII-A) and (IX-A), including thosepreferred aspects described above, m is 1 to 3.

Pharmaceutical Compositions

The present disclosure further provides a pharmaceutical composition fortreating a disease (e.g., cancer, such as a cancer associated withfolate receptor expressing tumor), wherein the composition comprises aneffective amount of a carrier particle-drug conjugate described herein,e.g., an NDC described herein.

In specific aspects of the present disclosure, the pharmaceuticalcomposition comprising the carrier particle drug conjugate (e.g., an NDCdescribed herein) can be used to treat cancer selected from the groupconsisting of ovarian cancer, endometrial cancer, fallopian tube cancer,cervical cancer, breast cancer, lung cancer, mesothelioma, uterinecancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer,rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer,kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia(CML). The pharmaceutical composition comprising the NDCs may also beused for targeting tumor associated macrophages, e.g., to modify theimmune status of a tumor in a subject.

The pharmaceutical compositions of the present disclosure may comprise apharmaceutically acceptable excipient, such as a non-toxic carrier,adjuvant, diluent, or vehicle that does not negatively impact thepharmacological activity of the carrier particle drug-conjugate (e.g.,NDC) with which it is formulated. Pharmaceutically acceptable excipientsuseful in the manufacture of the pharmaceutical compositions of thepresent disclosure are any of those that are well known in the art ofpharmaceutical formulation, and can include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Pharmaceutically acceptable excipientsuseful in the manufacture of the pharmaceutical compositions of thepresent disclosure include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins (e.g., human serumalbumin), buffer substances (e.g., phosphates), glycine, sorbic acid,potassium sorbate, glyceride mixtures (e.g., mixtures of saturatedvegetable fatty acids), water, salts or electrolytes (e.g., protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts), colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

The pharmaceutical compositions of the present disclosure may beadministered orally in the form of a suitable pharmaceutical unit dosageform. The pharmaceutical compositions of the present disclosure may beprepared in many forms that include tablets, hard or soft gelatincapsules, aqueous solutions, suspensions, liposomes, and otherslow-release formulations, such as shaped polymeric gels.

Suitable modes of administration for the carrier particle-drug conjugate(e.g., NDC) or composition thereof include, but are not limited to,oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic,subcutaneous, intramuscular, transdermal, spinal, intrathecal,intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphaticadministration, intra-tumoral, and other routes suitable for systemicdelivery of active ingredients.

The present pharmaceutical composition may be administered by any methodknown in the art, including, without limitation, transdermal (passivevia patch, gel, cream, ointment or iontophoretic); intravenous (bolus,infusion); subcutaneous (infusion, depot); transmucosal (buccal andsublingual, e.g., orodispersible tablets, wafers, film, and effervescentformulations); conjunctival (eyedrops); rectal (suppository, enema)); orintradermal (bolus, infusion, depot). The composition may be deliveredtopically.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The pharmaceutical compositions of the present disclosure may also beformulated for parenteral administration (e.g., by injection, forexample, bolus injection or continuous infusion) and may be presented inunit dosage form in ampules, pre-filled syringes, infusion containers(e.g., small volume infusion containers), or multi-dose containers, thatmay contain an added preservative.

The pharmaceutical compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulating agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the pharmaceutical compositions of the presentdisclosure may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration (e.g., to the epidermis), the pharmaceuticalcompositions may be formulated as ointments, creams or lotions, or asthe active ingredient of a transdermal patch. Suitable transdermaldelivery systems are disclosed, for example, in A. Fisher et al. (U.S.Pat. No. 4,788,603), and R. Bawa et al. (U.S. Pat. Nos. 4,931,279;4,668,506; and 4,713,224), each of which are incorporated herein byreference in their entireties. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, or coloring agents. The pharmaceuticalcompositions can also be delivered via ionophoresis, e.g., as disclosedin U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842, each of which areincorporated herein by reference in their entireties.

Pharmaceutical compositions suitable for topical administration in themouth include unit dosage forms such as lozenges comprising apharmaceutical composition of the present disclosure in a flavored base,such as sucrose and acacia or tragacanth; pastilles comprising thepharmaceutical composition in an inert base such as gelatin and glycerinor sucrose and acacia; mucoadherent gels, and mouthwashes comprising thepharmaceutical composition in a suitable liquid carrier.

For topical administration to the eye, the pharmaceutical compositionscan be administered as drops, gels (S. Chrai et al, U.S. Pat. No.4,255,415), gums (S. L. Lin et al, U.S. Pat. No. 4,136,177) or via aprolonged-release ocular insert (A. S. Michaels, U.S. Pat. No. 3,867,519and H. M. Haddad et al., U.S. Pat. No. 3,870,791), each of which areincorporated herein by reference in their entireties.

When desired, the above-described pharmaceutical compositions can beadapted to give sustained release of a therapeutic compound employed,e.g., by combination with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the pharmaceutical composition withthe softened or melted carrier(s) followed by chilling and shaping inmolds.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayscontaining, in addition to the nanoparticles and the therapeutic agent,such carriers are well known in the art.

For administration by inhalation, the pharmaceutical compositionsaccording to the present disclosure are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thepharmaceutical compositions of the present disclosure may take the formof a dry powder composition, for example, a powder mix of thepharmaceutical composition and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges or, e.g., gelatin or blister packsfrom which the powder may be administered with the aid of an inhalatoror insufflator.

For intra-nasal administration, the pharmaceutical compositions of thepresent disclosure may be administered via a liquid spray, such as via aplastic bottle atomizer. Typical of these are the Mistometer®(isoproterenol inhaler- Wintrop) and the Medihaler® (isoproterenolinhaler-Riker).

Pharmaceutical compositions of the present disclosure may also containother adjuvants such as flavorings, colorings, anti-microbial agents, orpreservatives.

It will be further appreciated that the amount of the pharmaceuticalcompositions suitable for use in treatment will vary not only with thetherapeutic agent selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or clinician. For evaluations of these factors, see J. F.Brien et al., Europ. J. Clin. Pharmacol, 14, 133 (1978); and Physicians'Desk Reference, Charles E. Baker, Jr., Pub., Medical Economics Co.,Oradell, N.J. (41′ ed., 1987), each of which are incorporated herein byreference in their entireties.

Administration and Methods of Treatment

Carrier particle-drug conjugates (e.g., NDCs) of the present disclosurecan be administered to a subject. The subject can be a mammal,preferably a human. Mammals include, but are not limited to, murines,rats, rabbits, simians, bovines, ovine, swine, canines, feline, farmanimals, sport animals, pets, equine, and primates.

Carrier particle-drug conjugates (e.g., NDCs) may be administered to asubject by, but not restricted to, the following routes: oral,intravenous, nasal, subcutaneous, local, intramuscular or transdermal.For example, the NDCs of the present disclosure may be administered to asubject intravenously.

The methods and compositions of the present disclosure can be used tohelp a physician or surgeon to identify and characterize areas ofdisease, such as cancers, including, but not restricted to, cancers thatoverexpress folate receptor (e.g., folate receptor alpha, or folatereceptor beta), to distinguish diseased and normal tissue, such asdetecting tumor margins that are difficult to detect using an ordinaryoperating microscope, e.g., in brain surgery, to help dictate atherapeutic or surgical intervention, e.g., by determining whether alesion is cancerous and should be removed or non-cancerous and leftalone, or in surgically staging a disease.

The methods and compositions of the present disclosure may be used, butare not limited to, metastatic disease detection, treatment responsemonitoring, and targeted delivery of payload, including by passing theblood-brain barrier.

The methods and compositions of the present disclosure can also be usedin the detection, characterization and/or determination of thelocalization of a disease, including early disease, the severity of adisease or a disease-associated condition, the staging of a disease,and/or monitoring a disease. The presence, absence, or level of anemitted signal can be indicative of a disease state.

The methods and compositions of the present disclosure can also be usedto monitor and/or guide various therapeutic interventions, such assurgical and catheter-based procedures, and monitoring drug therapy,including cell based therapies. The methods of the present disclosurecan also be used in prognosis of a disease or disease condition.Cellular subpopulations residing within or marginating the disease site,such as stem-like cells (“cancer stem cells”) and/orinflammatory/phagocytic cells may be identified and characterized usingthe methods and compositions of the present disclosure.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected or monitored (before, during orafter therapy) include cancer (for example, melanoma, thyroid,colorectal, ovarian, lung, breast, prostate, cervical, skin, brain,gastrointestinal, mouth, kidney, esophageal, bone cancer), that can beused to identify subjects that have an increased susceptibility fordeveloping cancer and/or malignancies, i.e., they are predisposed todevelop cancer and/or malignancies, inflammation (for example,inflammatory conditions induced by the presence of cancerous lesions),cardiovascular disease (for example, atherosclerosis and inflammatoryconditions of blood vessels, ischemia, stroke, thrombosis), dermatologicdisease (for example, Kaposi's Sarcoma, psoriasis), ophthalmic disease(for example, macular degeneration, diabetic retinopathy), infectiousdisease (for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome (AIDS)), immunologicdisease (for example, an autoimmune disorder, lymphoma, multiplesclerosis, rheumatoid arthritis, diabetes mellitus), central nervoussystem disease (for example, a neurodegenerative disease, such asParkinson's disease or Alzheimer's disease), inherited diseases,metabolic diseases, environmental diseases (for example, lead, mercuryand radioactive poisoning, skin cancer), bone-related disease (forexample, osteoporosis, primary and metastatic bone tumors,osteoarthritis) and a neurodegenerative disease.

The methods and compositions of the present disclosure, therefore, canbe used, for example, to determine the presence and/or localization oftumor and/or co-resident stem-like cells (“cancer stem cells”), thepresence and/or localization of inflammatory cells, including thepresence of activated macrophages, for instance in peritumoral regions,the presence and in localization of vascular disease including areas atrisk for acute occlusion (i.e., vulnerable plaques) in coronary andperipheral arteries, regions of expanding aneurysms, unstable plaque incarotid arteries, and ischemic areas. The methods and compositions ofthe present disclosure can also be used in identification and evaluationof cell death, injury, apoptosis, necrosis, hypoxia and angiogenesis(PCT/US2006/049222).

The methods of the present disclosure comprise administering to asubject in need thereof an effective amount of an NDC described herein.An “effective amount” is an amount of the carrier particle drugconjugate, e.g., NDC, that elicits a desired biological or medicinalresponse under the conditions of administration, such as an amount thatreduces the signs and/or symptoms of a disease or disorder beingtreated, e.g., reduces tumor size or tumor burden. The actual amountadministered can be determined by an ordinarily skilled clinician basedupon, for example, the subject's age, weight, sex, general heath andtolerance to drugs, severity of disease, dosage form selected, route ofadministration, and other factors

For example, the NDC can be administered to the subject in need thereofintravenously.

In specific aspects of the method, the subject has a cancer selectedfrom the group consisting of ovarian cancer, endometrial cancer,fallopian tube cancer, cervical cancer, breast cancer, lung cancer,mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML).

The present disclosure also includes use of carrier particle-drugconjugates (e.g., NDCs) for treating a folate receptor expressing tumor.For example, the use of the NDC may comprise administration to thesubject in need thereof intravenously.

The present disclosure also relates to the use of carrier particle-drugconjugates (e.g., NDCs) in a subject with cancer selected from the groupconsisting of ovarian cancer, endometrial cancer, fallopian tube cancer,cervical cancer, breast cancer, lung cancer, mesothelioma, uterinecancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer,rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer,kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia(CML).

The carrier particle-drug conjugates (e.g., NDCs) of the presentdisclosure may also be used in the manufacture of a medicament fortreating a folate receptor expressing tumor, wherein the carrierparticle-drug conjugate (e.g., NDC) is administered to the subject inneed thereof intravenously and wherein the subject has a cancer selectedfrom the group consisting of ovarian cancer, endometrial cancer,fallopian tube cancer, cervical cancer, breast cancer, lung cancer,mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML).

The compositions and methods disclosed herein can include compositionsand methods that include administering a carrier particle drug conjugate(e.g., NDC) as disclosed herein in combination with one or moreadditional anti-cancer agents. In such circumstances the carrierparticle drug conjugate (e.g., NDC) can be administered before,substantially concurrently with, or after the additional agent oragents. Suitable additional agents, include, for examplechemotherapeutic agents such as mechlorethamine, cyclophosphamide,melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea,carmustine, lomustine, semustine, fotemustine, streptozotocin,dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin,diaziquone, cisplatin, carboplatin, oxaliplatin, procarbazine,hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g.5-fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine,azacitidine, fludarabine, nelarabine, cladribine, clofarabine,pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine,vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, irinotecan,topotecan, camptothecin, etoposide, mitoxantrone, teniposide,novobiocin, merbarone, doxorubicin, daunorubicin, epirubicin,idarubicin, pirarubicin, aclarubicin, mitomycin C, actinomycin,bleomycin, bisantrene, gemcitabine, cytarabine, and the like. Otheranti-cancer agents that can be used with a carrier particle drugconjugate (e.g., NDC) in the compositions and methods disclosed hereininclude, immune check point inhibitors (e.g., anti-PD1, anti-PDL1,anti-CTLA4 antibodies), hormone receptor antagonists, otherchemotherapeutic conjugates (e.g., in the form of antibody-drugconjugates, nanoparticle drug conjugates, and the like), and the like.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe embodiments of the invention and the appended claims, the singularforms of “a”, “an” and “the” are intended to include the plural forms aswell, unless the context clearly indicates otherwise. Also, as usedherein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items.

The term “about,” when referring to a value means±20%, or ±10. Further,the term “about” when used in connection with one or more numbers ornumerical ranges, should be understood to refer to all such numbers,including all numbers in a range and modifies that range by extendingthe boundaries above and below the numerical values set forth. Therecitation of numerical ranges by endpoints includes all numbers, e.g.,whole integers, including fractions thereof, subsumed within that range(for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, aswell as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) andany range within that range.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

It will be understood that in the detailed description and appendedclaims, the abbreviations and nomenclature employed are those which arestandard in amino acid and peptide chemistry.

ABBREVIATIONS

The abbreviations used in this disclosure, unless otherwise indicatedare as follows:

-   Fmoc: Fluorenylmethoxycarbonyl-   MeOH: Methanol-   Cit-OH: L-Citrulline-   DCM: Dichloromethane-   EEDQ: 2-Ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline-   THF: Tetrahydrofuran-   NMR: Nuclear Magnetic Resonance-   DMSO: Dimethyl sulfoxide-   LCMS: Liquid Chromatography-Mass Spectrometry-   TEA: Triethylamine-   HATU:    (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium    3-oxide hexafluorophosphate-   DMF: Dimethylformamide-   DIPEA: N,N-Diisopropylethylamine-   TMSCN: Trimethylsilyl cyanide-   RP HPLC: Reverse Phase High-Pressure Liquid Chromatography-   SFC: Supercritical fluid chromatography-   CAN: Acetonitrile-   NMP: N-Methyl pyrrolidone-   r.t: Room Temperature-   TEA: Triethyl amine-   TFA: Trifluoroacetic acid-   MTBE: Methyl tert-butyl ether-   EtOAC: Ethyl acetate-   PyBOP:    (Benzotrizole-1-yl-oxytripyrrolidinenophosphoniumhexafluorophosphate)

Definitions

As used herein, the term “alkyl” refers to monovalent aliphatichydrocarbon group that may comprise 1 to 18 carbon atoms, such as 1 toabout 12 carbon atoms, or 1 to about 6 carbon atoms (“C₁₋₁₈ alkyl”). Analkyl group can be straight chain, branched chain, monocyclic moiety orpolycyclic moiety or combinations thereof. Examples of alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbomyl, and the like. Each instance of an alkyl group maybe independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents e.g., for instance from 1 to 5 substituents, 1 to 3substituents, or 1 substituent.

As used herein, the term “alkenyl” refers to a monovalent straight-chainor branched hydrocarbon group having from 2 to 18 carbon atoms, one ormore carbon-carbon double bonds, and no triple bonds (“C₂₋₁₈ isalkenyl”). An alkenyl group may have 2 to 8 carbon atoms, 2 to 6 carbonatoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms.The one or more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of alkenylgroups include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenylbutadienyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl,octatrienyl, and the like, Each instance of an alkenyl group may beindependently optionally substituted i.e., unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents e.g., for instance from 1 to 5 substituents. 1to 3 substituents, or 1 substituent.

As used herein, the term “alkynyl” refers to a monovalent straight-chainor branched hydrocarbon group having from 2 to 18 carbon atoms, one ormore carbon-carbon triple bonds “C₂₋₁₈ alkynyl”). The alkynyl group nayhave 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to4 carbon atoms, or 2 to 3 carbon atoms. The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of alkynyl groups include ethynyl, 1-propynyl,2-propynyl. 1-butynyl, 2-butynyl, and the like. Each instance of analkynyl group may be independently optionally substituted, i.e.,unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents, e.g., for instancefrom 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

As used herein, the term “heteroalkyl” refers to a non-cyclic stablestraight or branched chain, or combinations thereof, including at leastone carbon atom and at least one heteroatom selected from the groupconsisting of O, N, P, Si, and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quatemized. The heteroatom(s) O, N, P, S, and Si may beplaced at any position of the heteroalkyl group.

The terms “alkylene,” “alkenylene,” “alkynylene,” or “heteroalkylene,”alone or as part of another substituent, mean, unless otherwise stated,a divalent radical derived from an alkyl, alkenyl, alkynyl, orheteroalkyl, respectively. The term “alkenylene,” by itself or as partof another substituent, means, unless otherwise stated, a divalentradical derived from an alkene. An alkylene, alkenylene, alkynylene, orheteroalkylene group may be described as, e.g., a C₁₋₆-memberedalkylene, C₁₋₆-membered alkenylene, C₁₋₆-membered alkynylene, orC₁₋₆-membered heteroalkylene, wherein the term “membered” refers to thenon-hydrogen atoms within the moiety. In the case of heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— may represent both —C(O)₂R′— and —R′C(O)₂—. Eachinstance of an alkylene, alkenylene, alkynylene, or heteroalkylene groupmay be independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkylene”) or substituted (a “substitutedheteroalkylene”) with one or more substituents.

As used herein, the terms “substituted alkyl,” “substituted alkenyl,”“substituted alkynyl,” “substituted heteroalkyl,” “substitutedheteroalkenyl,” “substituted heteroalkynyl,” “substituted cycloalkyl,”“substituted heterocyclyl,” “substituted aryl,” and “substitutedheteroaryl” refer to alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl moieties, respectively, having substituents replacing one ormore hydrogen atoms on one or more carbons or heteroatoms of the moiety.Such substituents can include, for example, alkyl, alkenyl, alkynyl,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above.

As used herein, the term “alkoxy” refers to a group of formula —O-alkyl.The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

As used herein, the term “aryl,” refers to stable aromatic ring system,that may be monocyclic or polycyclic, of which all the ring atoms arecarbon, and which may be substituted or unsubstituted. The aromatic ringsystem may have, for example, 3-7 ring atoms. Examples include phenyl,benzyl, naphthyl, anthracyl, and the like. Each instance of an arylgroup may be independently optionally substituted, i.e., unsubstituted(an “unsubstituted aryl”) or substituted (a “substituted aryl”) with oneor more substituents.

As used herein, the term “heteroaryl” refers to an aryl group thatincludes one or more ring heteroatoms. For example, a heteroaryl caninclude a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, or9-membered bicyclic aromatic heterocyclic ring which consists of carbonatoms and one or more heteroatoms, independently selected from the groupconsisting of nitrogen, oxygen and sulfur. The nitrogen atom may besubstituted or unsubstituted (e.g., N or NR₄ wherein R₄ is H or othersubstituents, as defined). Examples of heteroaryl groups includepyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, pyrimidine, and the like.

As used herein, the terms “cycloalkylene,” “heterocyclylene,” “arylene,”and “heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from a cycloalkyl, heterocyclyl, aryl, andheteroaryl, respectively. Each instance of a cycloalkylene,heterocyclylene, arylene, or heteroarylene may be independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted arylene”)or substituted (a “substituted heteroarylene”) with one or moresubstituents.

As used herein, the term “cycloalkyl”, is intended to includenon-aromatic cyclic hydrocarbon rings, such as hydrocarbon rings havingfrom three to eight carbon atoms in their ring structure. Cycloalkyl caninclude cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl and the like.The cycloalkyl group can be either monocyclic (“monocyclic cycloalkyl”)or contain a fused, bridged or spiro ring system such as a bicyclicsystem (“bicyclic cycloalkyl”) and can be saturated or can be partiallyunsaturated. “Cycloalkyl” also includes ring systems wherein thecycloalkyl ring, as defined above, is fused with one or more aryl groupswherein the point of attachment is on the cycloalkyl ring, and in suchinstances, the number of carbons continue to designate the number ofcarbons in the cycloalkyl ring system. Each instance of a cycloalkylgroup may be independently optionally substituted, i.e., unsubstituted(an “unsubstituted cycloalkyl”) or substituted (a “substitutedcycloalkyl”) with one or more substituents.

As used herein, the term “heterocyclyl” refers to a monovalent cyclicmolecular structure comprising atoms of at least two different elementsin the ring or rings (i.e., a radical of a heterocyclic ring).Additional reference is made to: Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997 as evidencethat heterocyclic ring is a term well-established in field of organicchemistry.

As used herein, the term “dipeptide” refers to a peptide that iscomposed of two amino-acid residues, that may be denoted herein as-A₁-A₂-. For example, dipeptides employed in the synthesis ofprotease-cleavable linker-payload conjugates of the present disclosuremay be selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys,Asp-Lys, Val- Lys, and Val-Ala.

As used herein, the term “functionalized polyethylene glycol” refers tothe polyethylene glycol comprising a functional group. For example, afunctionalized polyethylene glycol may be polyethylene glycolfunctionalized with a terminal group selected from the group consistingof azide,

wherein R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl. In preferred aspects,R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence ishydrogen. In preferred aspects, R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is methyl.

In some aspects of the present disclosure, the term “functionalizedpolyethylene glycol” refers to, but is not limited to the followingstructures.

As used herein, T₁ may refer to a functionalized polyethylene glycol ora C₅-C₆ alkyl chain that has a terminal group selected from the groupconsisting of azide,

wherein R^(1′), R²′ R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl. In preferred aspects ofT₁, R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence ishydrogen. In preferred aspects of T₁, R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is methyl. In preferred aspects, T₁ is afunctionalized polyethylene glycol that has an azide terminal group. Inpreferred aspects, T₁ is a C₅-C₆ alkyl chain that has an azide terminalgroup.

The repeat unit (—O—CH₂—CH₂—) of polyethylene glycol (PEG) can rangefrom 5-20 units, preferably 5-15 units and more preferably 6-12.

As used herein, T₁ may refer to a C₅-C₆ alkyl chain that has a terminalgroup selected from the group consisting of azide,

wherein R¹, R^(3′), R⁴‘ and R’ in each occurrence is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl or substituted orunsubstituted C₁₋₆ cycloalkyl. In preferred aspects, R^(1′), R^(2′),R^(3′), R^(4′) and R^(5′) in each occurrence is hydrogen. In preferredaspects, R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence ismethyl.

Monofunctionalized azide-terminated PEG and monofunctionalizedazide-terminated C₅-C₆ alkyl chain can be made from PEG using knownprocedures and suitable reagents, such as those disclosed in the Schemesprovided herein.

As used herein, the term “halo” or “halogen” refers to F, Cl, Br, or I.

An aryl or heteroaryl group described herein can be substituted at oneor more ring positions with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

As used herein, the term “hydroxy” refers to a group of formula —OH.

As used herein, the term “hydroxyl” refers to a hydroxyl radical (·OH).

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. In general, the term “substituted” means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound. The term “substituted” caninclude substitution with all permissible substituents of organiccompounds, such as any of the substituents described herein that resultin the formation of a stable compound. For purposes of the presentdisclosure, heteroatoms such as nitrogen may have hydrogen substituentsand/or any suitable substituent as described herein which satisfy thevalencies of the heteroatoms and results in the formation of a stablemoiety.

As used herein, a “targeting ligand” is a molecule that can be bonded toa carrier particle (e.g., nanoparticle) and target the carrier particleto a tumor or cancer cell, typically by binding to the tumor or cancercell (such as by binding to a protein expressed on the surface of thetumor or cancer cell). The targeting ligand can be any suitable moleculesuch as a small organic molecule (e.g., folate or a folate analog), anantigen-binding portion of an antibody (e.g. a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, a scFv fragment, a Fv fragment, a dsFvdiabody, a dAb fragment, a Fd′ fragment, a Fd fragment, or an isolatedcomplementarity determining region (CDR) region), an antibody mimetic(e.g., an aptamer, affibody, affilin, affimer, anticalin, avimer,Darpin, and the like), the binding domain of a receptor, a nucleic acid,lipid and the like.

As used herein, the term “tetrapeptide” refers to a peptide that iscomposed of four amino-acid residues, that may be denoted herein as-A₁-A₂-A₃-A₄-. Tetrapeptides employed in the synthesis ofprotease-cleavable linker-payload conjugates of the present disclosureis selected from the group consisting of Val-Phe-Gly-Sar (SEQ ID NO: 8),Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10), Val-Ala-Gly-Sar (SEQID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12), Val-Cit-Gly-Pro,Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQ ID NO: 15),Val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Val-Lys-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, Val-Phe-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Ala-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Phe-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, and Trp-Lys-Gly-any natural orunnatural N-alkyl substituted alpha amino acid.

Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. As used herein, the term “protecting group” refers toa particular functional moiety, e.g., O, S, or N, that is temporarilyblocked so that a reaction can be carried out selectively at anotherreactive site in a multifunctional compound. Protecting groups may beintroduced and removed at appropriate stages during the synthesis of acompound using methods that are known to one of ordinary skill in theart. The protecting groups are applied according to standard methods oforganic synthesis as described in the literature (Theodora W. Greene andPeter G. M. Wuts (2007) Protecting Groups in Organic Synthesis, 4thedition, John Wiley and Sons, incorporated by reference with respect toprotecting groups).

Exemplary protecting groups include, but are not limited to, oxygen,sulfur, nitrogen and carbon protecting groups. For example, oxygenprotecting groups include, but are not limited to, methyl ethers,substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM(pimethoxybenzyloxymethyl ether), optionally substituted ethyl ethers,optionally substituted benzyl ethers, silyl ethers (e.g., TMS(trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate,acetate, benzoate (Bz), trifluoroacetate, dichloroacetate) carbonates,cyclic acetals and ketals. In addition, nitrogen protecting groupsinclude, but are not limited to, carbamates (including methyl, ethyl andsubstituted ethyl carbamates (e.g., Troc), amides, cyclic imidederivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enaminederivatives, etc. Amino protecting groups include, but are not limitedto fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc),carboxybenzyl (Cbz), acetamide, trifluoroacetamide, etc. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present disclosure is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups may be utilized according to methods known to oneskilled in the art.

Throughout this disclosure, a nanoparticle-drug-conjugate (NDC) maysometimes be referred to as a CDC (C′Dot-drug-conjugate), e.g., aFA-CDC.

The following examples are provided to further illustrate theembodiments of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Examples

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. These examples areoffered to illustrate the nanoparticle drug conjugates, methods of use,and methods of making, and are not to be construed in any way aslimiting their scope.

The compounds provided herein can be prepared from readily availablestarting materials using modifications to the specific synthesisprotocols set forth below that would be well known to those of skill inthe art. It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvents used, butsuch conditions can be determined by those skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice ofsuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in Greene et al. Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

General Methods

Methods useful for making the compounds discussed herein are set forthin the following examples, and are generalized here. One of skill in theart will recognize that these examples can be adapted to prepare thelinker-payload conjugates, linkers and payloads and theirpharmaceutically accepted salts thereof according to the presentdisclosure. In the reactions described, reactive functional groups, suchas hydroxy, amino, imino, thio or carboxy groups, may be protectedwherever desired, e.g., to avoid unwanted reactions. Conventionalprotecting groups may also be used in accordance with standard practiceand techniques of synthesis. The materials needed to synthesize thenovel linkers bearing payloads such as exatecan mesylate (16), or SN-38(34) were obtained commercially, and their corresponding analogs areprepared as disclosed in the following Examples, e.g., Examples 76-87.

Example 1: Synthesis of Functionalized Polyethylene Glycol with ReactiveTerminal Groups

In Formulae (I-A)-(X-A), T¹ is a functionalized polyethylene glycol or aC₅-C₆ alkyl chain that has a terminal group selected from the groupconsisting of azide,

that facilitates the conjugation of the linker-payload conjugates to atargeting group such an antibody or a carrier such as silicananoparticles, C-DOTS, etc.

Functionalized polyethylene glycol or a C₅-C₆ alkyl chain with an azidemoiety can be reacted with the targeting group or the carrier havingacetylenic or olefinic functional groups via “click” chemistry.Similarly, if the functionalized polyethylene glycol or a C₅-C₆ alkylchain having a nitrone, nitrile oxide or diene as terminal groups can beattached to the targeting group or the carrier having an olefinic bondor acetylenic bond through a (4+2) or (3+2) cycloaddition reaction. Onthe other hand, if the functionalized polyethylene glycol or a C₅-C₆alkyl chain having a —CO—O-succinimide group can be attached to thenanoparticle or the targeting moiety such as an antibody through the—NH₂ group or —SH group present on their surface.

The functionalized polyethylene glycol containing reactive terminalgroups such as azide, nitrone, nitrile oxide or diene can be prepared asoutlined in Schemes 1a-c respectively.

The functionalized polyethylene glycol can be prepared from PEGacid-t-butyl ester (1) as the starting material. Scheme 1a depicts thesynthesis of functionalized polyethylene glycol with terminal nitrileoxide group (2). Compound (1) can be oxidized to the aldehyde derivative(1a) using Swern Oxidation or using Dess-Martin reagent (DMP). Thepivotal aldehyde compound (1a) can be reacted with hydroxyl aminehydrochloride to form an oxime, which can be converted to the nitrileoxide using a two-step procedure as indicated in Scheme 1a.

The aldehyde (1a) can also be converted to a functionalized polyethyleneglycol with terminal nitrone group (3) (Scheme 1b) and a functionalizedpolyethylene glycol with terminal diene (4) (Scheme 1c) by the sequenceof the reaction given in Schemes 1b and 1c respectively. The t-butylgroup can be removed under acidic condition using hydrochloric acid ortrifluoro acetic acid and can be reacted with an amino group on thelinker or linker-payload conjugates under standard peptide couplingconditions.

Example 2: Synthesis of Protease-Cleavable Linker-Payload ConjugatesContaining Dipeptides

Scheme 2 provides the general method of preparation of severalprotease-cleavable linker-payload conjugates such as, but not limited toExamples 18, 24 and 29 respectively. -A₁-A₂- in Scheme 2 refers todipeptides that can be selected from a group consisting of Val-Cit,Phe-Lys, Trp- Lys, Asp-Lys, Val- Lys, and Val-Ala.

An appropriately substituted 1-(4-nitrophenyl) ethan-1-one (5), in amixture of water and aprotic polar solvent such as 1, 4-dioxane/H₂O wasoxidized with SeO₂ and Yb(OTf)₃ under inert atmosphere at r.t for 1 to 2hrs followed by heating the reaction mixture to 110° C. for 16 h. Aftercompletion of starting material, reaction mixture was cooled to ambienttemperature, quenched with ice cold water and extracted with EtOAc ordichloroethane. The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to get appropriatelysubstituted 2-hydroxy-2-(4-nitrophenyl) acetic acid (6) derivative. Thesubsequent transformations were carried out without purification.

The conversion of (5) to (6) was performed in solvent such as isopropylalcohol-THF mixture. In another embodiment, the reaction was performedat about 100 to about 150° C. In another embodiment, the reaction wasperformed in a sealed tube. The carboxylic acid (6) obtained wasesterified with either methanol or ethanol under standard Fischeresterification condition. The resultant, product (7) was then reactedwith an excess of palladium hydroxide to provide compound (8). In oneembodiment, the reaction was performed in a parr shaker or an autoclave.In another embodiment, the reaction was performed at about 60 psi ofhydrogen pressure. The amino group of the compound (8) reacted with thenatural or unnatural, Fmoc protected aminoacids (A₁-Fmoc) under standardpeptide coupling conditions either using EEDQ at room temperature inpolar aprotic solvent solvents (DMF or THF) or HOAT/EDC. HCl in DMF. Theresultant product (9) was deprotected with Piperidine or any Sec.organic base in DMF or THF, to deprotect the —NH₂ group and to provideproduct (10). Following the same peptide coupling protocol, anotheramino acid can be reacted with (10) and subsequent deprotection of theFmoc group can yield (12). Following the above mentioned sequence ofpeptide coupling and deprotection strategy several amino acid chains canbe incorporated.

Scheme 2 provides a general scheme to incorporate two amino acidstowards preparation of compound (12). Compound (12) can be reacted witha PEG carboxylic acid bearing the appropriate functional group (such asan azide group, as shown for example) using coupling agents such asHOBT/EDC. Subsequently, compound (14) was hydrolyzed with aqueous LiOHin THF for 1 hr. The resulting carboxylic acid intermediate (15) can becoupled with a payload with an amino functional group such as Exatecan(16) to give the linker-payload conjugate (16-A).

Example 3: Synthesis of Protease-Cleavable Linker-Payload ConjugatesContaining Tetrapeptides

Scheme 3 provides the general method of preparation of severalprotease-cleavable linker-payload conjugates such as, but not limited toExamples 9, 10, 11, 15, 16, 17, 25, 26, 27, 28, 30, 31, 32, 33, 34, 37,39, 43, 45, 52 and 53 respectively. -A₁-A₂-A₃-A₄-in Scheme 3 refer totetrapeptides that can be selected from a group consisting ofVal-Phe-Gly-Sar (SEQ ID NO: 8), Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ IDNO: 10), Val-Ala-Gly-Sar (SEQ ID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO:12), Val-Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro(SEQ ID NO: 15), Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino.

To an appropriately substituted (4-aminophenyl)methanol (17) in DMF wasreacted with imidazole and tert-butyl(chloro)diphenylsilane at 0° C. toyield (18). The appropriately substituted4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (18) was reacted withthe amino acid sequences using the regular peptide coupling protocol.The amino acids that are used are appropriately protected. Example, theamino group is protected as the Fmoc group. Compound (18) can be reactedwith a Fmoc protected amino acids in the presence of coupling agentssuch as HATU and base such as DIEA in a polar aprotic solvent such asDMF. In order to incorporate another amino acid, the Fmoc group inintermediate (19) can be deprotected using a sec. organic base. Similarsequence of reaction was repeated with Fmoc protected amino acids.Subsequently the Fmoc group was removed and the N-terminus was reactedwith the appropriately functionalized PEG carboxylic acid (13) to yield(24). The t-butyl-dimethyl-silyl group was removed by reacting compound(24) with NH₄F in methanol at room temperature. In order to connect thelinker with the Exatecan (16), a carbonate ester (26) was prepared using4-nitro-phenyl chloroformate (25) by reacting (25a) in DCM/pyridine at0° C. In some examples, these intermediates can be isolated by columnchromatography. The carbonate finally reacted with payload containing anamino functional group such as Exatecan or SN-38 or their correspondinganalog to yield (16-B). The same sequence of reaction can be carriedout, where in X₁ and X₂ are independently —CH— or —N—.

Example 4: Synthesis of Protease-Cleavable Linker-Payload ConjugatesContaining Tetrapeptides

Scheme 4 provides the general method of preparation of severalprotease-cleavable linker-payload conjugates such as, but not limited toExamples 12, 13, 14, 19 and 20 respectively. In these Examples, Y₁ is—COO—C₁-C₆ alkyl or —CONR¹R² and the rest of the variables are asdefined. -A₁-A₂-A₃-A₄- in Scheme 4 refer to tetrapeptides that can beselected from a group consisting of Val-Phe-Gly-Sar (SEQ ID NO: 8),Val-Cit-Gly-Sar, Val-Lys-Gly-Sar (SEQ ID NO: 10), Val-Ala-Gly-Sar (SEQID NO: 11), Val-Phe-Gly-Pro (SEQ ID NO: 12), Val-Cit-Gly-Pro,Val-Lys-Gly-Pro (SEQ ID NO: 14), Val-Ala-Gly-Pro (SEQ ID NO: 15),Val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Val-Lys-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, Val-Phe-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Val-Ala-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Phe-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, and Trp-Lys-Gly-any natural orunnatural N-alkyl substituted alpha amino.

The appropriately substituted 4-nitro-benzaldehyde (27) can be reactedwith ZnI/TMS-CN in an organic solvent such as dichloromethane ordichloroethane at room temperature. The resultant cyanohydrine (28) washydrolyzed to the carboxylic acid using mineral acids such as sulfuricacid and subsequently esterified under Fisher esterification condition.The nitro group in compound (30) was converted to the amino byhydrogenating the compound (30) with 10% Pd/C in either MeOH or ethanol.The amino group in compound (31) can be reacted with the appropriateamino acids, using standard coupling agents such as EEDQ in achlorinated solvent as described in the Schemes 2 and 3. Theintermediate (32) was reacted with thionyl chloride at room temperaturein THF as solvent. The chloro derivative (33) can be reacted with a payload containing hydroxyl functionality or an amino functionality(Examples 52 and 53) using anhydrous Cs₂CO₃ or K₂CO₃ in DMF at roomtemperature. After the incorporation of the payload (in this case, SN-38(34)), the intermediate (35) can be converted to the final compound (37)by the same sequence of reactions, as described in Schemes 2 and 3. Asimilar sequence of reaction depicted in Scheme 4 can be adopted tosynthesize compounds of Formula II. It is understood that the finallinker-payload conjugates can exist as a mixture of diastereomers andthey can be separated using reverse phase HPLC. The conditions for thepurification are described herein.

In a linker-payload conjugate containing a tetrapeptide such asVal-Lys-Gly-Sar, the Payload can be released by a two-step mechanism asoutlined in Scheme 4a.

Example 5: Synthesis of Protease-Cleavable Linker-Payload ConjugatesContaining Pyrrole or Imidazole Groups

Scheme 5 provides the general method of preparation of severalprotease-cleavable linker-payload conjugates containing substitutedpyrrole or imidazoles groups at Y, such as, but not limited to Examples38 and 44.

Appropriately substituted 4-amino-benzylalcohol derivative (17) wasreacted with appropriate F-moc protected amino acids by the conventionalpeptide coupling reaction to yield (50). The hydroxy group in the benzylalcohol group can be converted to an iodo group by reacting compound(50) with NaI/TMSCl in acetonitrile at room temperature to yield (51).The iodo compound (51) was reacted with the appropriately substitutedpyrrole or imidazole carboxylic acid ester derivative or thecorresponding alcohol derivative (52). Subsequently, compound (52) wasdeprotected using a sec. organic base such as piperidine. The aminogroup in compound (52) was reacted with an another required amino acidand this process (coupling of the amino acid containing Fmoc anddeprotection of the Fmoc group) in a repetitive manner as outlinedbefore to yield a dipeptide or tetrapeptide bearing linker (55). Theappropriately reactive group substituted PEG-carboxylic acid (13) wascoupled with the amino terminal of the amino acid and can used toconjugate with a nanoparticle, antibody drug conjugates or to anacetylene or olefin containing moiety. If the pyrrole or imidazolemoiety bears a carboxylic acid, the ester group was hydrolyzed andcoupled with the amino containing payloads. Here in, Exatecan (16) isgiven as an example to yield compound (59). However, if derivative (56)is an alcohol (—CH₂OH) moiety, it was converted to an active ester byreacting with 4-nitrophenyl chloroformate to yield the active ester(57), which can be reacted with an amino containing payload such asExatecan (16) to yield compounds of general formula (58).

Example 6: Synthesis of Redox-sensitive Linker-payload Conjugates

Scheme 6 provides the general method of preparation of redox-sensitivelinker-payload conjugates such as, but not limited to Examples 57, 58,59, 61, 54, 55 and 56.

An appropriately substituted, 4-mercapto-benzoic acid (38) wasacetylated and converted to corresponding appropriately substituted(S-(4-(hydroxymethyl)phenyl) ethanethioate intermediate (39). Thecarboxylic acid was reduced using BH₃:THF complex at −10° C. to 0° C. toyield (40). The disulfide derivative with an amino substitutedderivative (45) was prepared by reacting compound (40) with thecommercially available reagent (41). The intermediate compound (42) wasconverted to the intermediate (45) by a two-step process as shown inScheme 6. An activated ester (46) was prepared by reacting compound (45)with 4-nitrophenyl chloroformate in the presence of organic base such astriethyl amine or DIEA. In some examples, the carbonate ester (46) canbe isolated and purified. The carbonate ester can be used to react witha payload having an amino group, which is depicted in Scheme 5.

Example 7: Synthesis of pH-Sensitive Linker-Payload Conjugates

Scheme 7 provides the general method of preparation of pH-sensitivelinker-payload conjugates such as, but not limited to Examples 64-75.

For the pH mediated linkers, the payload is initially converted intoclosely related derivatives such as (63) or (70). If the payloadcontains hydroxyl functionality such as (60), it was converted toderivative (62) by reacting derivative (60) with tert-butylchloroacetate (61) in the presence of base such as K₂CO₃ or Cs₂CO₃ inacetone or DMF. tert-butyl group was removed using acid such as TFA andcoupled with t-Boc protected hydrazine using standard coupling reagentsuch as EDC to give the hydrazide (63). Finally, compound (63) wascondensed with the ketone derivative (64) to yield the payload attachedto pH sensitive linker (65). Similarly if the payload contains an —NH₂group (66), a beta-keto ester (67) can be used to prepare a payloadconjugated pH sensitive linker (71) as depicted in Scheme 7.Accordingly, the —NH₂ group of the payload (66) was reacted with anappropriately substituted beta-keto ester derivative such as (67) toyield the intermediate (68). Intermediate (68) can be converted to thehydrazide derivative (70) by a two-step process as indicated in Example7, which can be condensed with the ketone derivative (64). R¹ and R² inScheme 7 can be selected from a group consisting of hydrogen,substituted or unsubstituted C₁₋₆ alkyl or substituted or unsubstitutedC₁₋₆ cycloalkyl.

Example 8: Synthesis of Semicarbazide pH-Sensitive Linker-PayloadConjugates

Scheme 8 provides the general method of preparation of semicarbazidepH-sensitive linker-payload conjugates such as, but not limited toExample 74.

Scheme 8 provides the general method of preparation of a semicarbazidepH-sensitive linker, wherein the payload containing the amino group isconverted to a semicarbazide derivative (73), by a two-step process. Thesemicarbazide (73) can be condensed with the appropriately substitutedketone derivative (64) to yield the pH-sensitive semicarbazone ofgeneral formula (74).

General Experimental Procedures:

Reagents were purchased from commercial suppliers(Combi-Blocks/SIGMA-ALDRICH) and used without further purification. Allnon-aqueous reactions were run in flame-dried glassware under a positivepressure of argon. Anhydrous solvents were purchased from commercialsuppliers (RANKEM). All the amino acids such as Cit, Val, Phe, Lys, Trp,Asp are naturally occurring amino acids with S-configuration. In severalexamples, tetrapeptide and unnatural amino acids can also be used. Flashchromatography was performed on 230-400 mesh silica gel with theindicated solvent systems. Proton Nuclear magnetic resonance spectrawere recorded on Bruker Spectrometer at 400 MHZ using DMSO as solvent.Peak positions are given in parts per million downfield fromtetramethylsilane as the internal standard. J values are expressed inhertz. Mass analyses were performed on (Agilent/Shimadzu) spectrometerusing electrospray (ES) technique. HPLC analyses were performed on(Agilent/Waters), PDA-UV detector equipped with a Gemini C-18 (1000×4.6mm; 5u) and all compounds tested were determined to be >95% pure usingthis method. As can be seen in many protease-cleavable linker-payloadconjugates, two peaks were isolated at the end of the reaction. ThePeak-A (or Peak-1) is the desired compound with the stereochemistry asshown. Compounds prepared according to the procedures described hereinmay be isolated by preparative HPLC methods. Representative HPLCconditions and methods are provided below:

Agilent UPLC-MS; Column: Column-YMC Triart C₁₈ (2.1×33 mm, 3u)

Gradient Conditions: Flow rate: 1.0 ml/min; column temperature: 50° C.;Solvent A: 0.01% HCOOH in water and Solvent B: 0.01% HCOOH in CH₃CN;Mobile phase: 95% [0.01% HCOOH in water] and 5% [0.01% HCOOH in CH₃CN]held for 0.50 min, then to 1% [0.01% HCOOH in water] and 99% [0.01%HCOOH in CH₃CN] in 3.00 min, held this conditions up to 4.00 min andfinally back to initial condition in 4.10 min and held for 4.50 min(Table 1).

TABLE 1 HPLC Gradient Conditions. TIME MODULE % A % B 0.00 Pumps 95 50.50 Pumps 95 5 3.00 Pumps 1 99 4.00 Pumps 1 99 4.10 Pumps 95 5 4.50Pumps 95 5

Example 9: Synthesis of4-((20S,23S)-1-azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(85)

(9H-fluoren-9-yl)methyl-(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-reidopentan-2-yl)carbamate(77): (4-aminophenyl) methanol (75) (6 g, 4.87 mmol), Fmoc-Cit-OH (76)(23.24 g, 58.54 mmol), EEDQ (36.15 g, 146.34 mmol) were mixed in DCM-THF(1:1) (600 ml) and stirred at ambient temperature under nitrogen for 16h. The reaction mixture was reduced to dryness under vacuum and purifiedby column chromatography on a silica cartridge eluting with methanol/DCMgradient (5-10%). The solvent was evaporated under vacuum to obtain 11 g(45%) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamatedesiredproduct (77) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.97 (s, 1H),7.90 (d, 2H), 7.73-7.76 (q, 2H), 7.65-7.67 (d, 1H), 7.54-7.56 (d, 2H),7.39-7.43 (t, 2H), 7.30-7.34 (m, 2H), 7.22-7.24 (d, 2H), 5.97-5.99 (t,1H), 5.41 (s, 1H), 5.07-5.10 (t, 1H), 4.42-4.43 (d, 2H), 4.14-4.13 (m,4H), 4.07-4.11 (q, 1H), 3.16-3.17 (d, 3H), 2.94-3.04 (m, 2H), 1.59-1.68(m, 2H), 1.38-1.47 (m, 2H). LCMS: MH⁺ 503, retention time 2.91 min.

(S)-2-amino-N-(4-(hydroxymethyl) phenyl)-5-ureidopentanamide (78): Asolution of(9H-fluoren-9-yl)-methyl-(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)(77) (10 g, 19.91 mmol) in DMF (30 ml) was treated with piperidine (5.9ml, 59.74 mmol) and the reaction mixture stirred at 0° C. under nitrogenatmosphere for 1 h. The reaction mixture was reduced to dryness undervacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (10-15%). The solvent was evaporatedunder vacuum to get 3.5 g (63%) of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (78) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 9.79 (s, 1H), 7.56-7.58 (d, 2H), 7.21-7.23 (d, 2H), 5.92-5.97(t, 1H), 5.35 (s, 1H), 5.06-5.09 (t, 1H), 4.41-4.43 (d, 2H), 3.27-3.38(m, 2H), 2.95-2.97 (d, 2H), 2.94-3.04 (m, 2H), 1.39-1.60 (m, 4H). LCMS:MH⁺ 281, retention time 1.04 min.

(9H-fluoren-9-yl)methyl-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (80):(S)-2-amino-N-(4-(hydroxymethyl) phenyl)-5-ureidopentanamide (78) (3.5g, 12.49 mmol), Fmoc-Val-OH (79) (5.08 g, 14.99 mmol), EEDQ (9.27 g,37.48 mmol) were mixed in DCM:THF (1:1) (300 ml) and was stirred atambient temperature under nitrogen atmosphere for 16 h. The reactionmixture was reduced to dryness under vacuum and purified by columnchromatography on a silica cartridge eluting with methanol/DCM gradient(5-10%). The solvent was evaporated under vacuum to get 5.8 g (77%) of(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(80) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.82 (s, 1H),8.34-8.36 (d, 1H), 7.86-7.89 (q, 2H), 7.69-7.75 (d, 2H), 7.55-7.57 (d,2H), 7.50-7.52 (d, 1H), 7.37-7.42 (q, 3H), 7.29-7.32 (q, 2H), 7.22-7.24(d, 2H), 5.95-5.97 (t, 1H), 5.39 (s, 2H), 5.09 (s, 1H), 4.41 (s, 3H),4.20-4.27 (m, 3H), 3.90-3.97 (t, 1H), 2.94-3.00 (m, 2H), 1.73-1.96 (m,3H), 1.14-1.58 (m, 3H), 0.83-0.89 (m, 6H). LCMS: MH⁺ 602, retention time2.87 min.

(9H-fluoren-9-yl)methyl-((S)-3-methyl-1-(((S)-1-((4-((((4-Nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(81):(9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(hydroxymethyl)-phenyl)-amino)-1-oxo-5-ureidopentan-2-yl)-amino)-3-methyl-1-oxobutan-2-yl)carbamate (80) (1 g, 1.66mmol), 4-nitrophenyl chloroformate (25) (1 g, 4.98 mmol) were treatedwith pyridine (0.52 g, 6.65 mmol) and the reaction mixture was stirredat ambient temperature under nitrogen atmosphere for 4 h. The reactionmixture was reduced to dryness under vacuum and purified by columnchromatography on a silica cartridge eluting with methanol/DCM gradient(2%). The solvent was evaporated under vacuum to get 400 mg (31%) of(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(81) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H),8.90-8.91 (d, 5H), 8.52-8.56 (t, 3H), 8.38-8.39 (d, 1H), 8.29-8.31 (d,2H), 8.01-8.04 (t, 5H), 7.86 (s, 2H), 7.67-7.74 (m, 3H), 7.49-7.57 (m,2H), 7.37-7.39 (d, 4H), 7.32 (s, 2H), 5.23 (s, 2H), 4.41 (s, 1H),4.21-4.25 (d, 4H), 3.94 (s, 2H), 2.96-2.98 (d, 2H), 1.97 (s, 1H), 1.75(s, 1H), 1.60 (s, 1H), 1.37-1.45 (d, 2H). LCMS: MH⁺767, retention time3.40 min.

(9H-fluoren-9-yl)methyl-((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamoyl)-oxy)-methyl)-phenyl)-amino)-1-oxo-5-ureidopentan-2-yl)-amino)-3-methyl-1-oxobutan-2-yl)-carbamate(82): Exatecan mesylate (16) (400 mg, 0.75 mmol),(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(81) (692.41 mg, 0.90 mmol) and TEA (0.20 ml, 1.51 mmol) were mixed inDMSO (15 ml) and stirred the reaction mixture at ambient temperatureunder nitrogen for 4 h. Ice cold water was added to the reaction mixtureand solid precipitation was observed. The reaction mixture was filteredand the residue was purified by column chromatography on a silicacartridge eluting with MeOH/DCM gradient (2-5%) to get 360 mg (45%) of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(82) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H),8.06-8.11 (m, 1H), 7.87-7.88 (d, 1H), 7.71-7.77 (m, 2H), 7.59-7.61 (d,1H), 7.50 (s, 1H), 7.31-7.40 (m, 5H), 6.51 (s, 1H), 5.96 (s, 1H),5.36-5.39 (d, 2H), 5.28 (s, 2H), 5.08 (s, 1H), 4.22-4.27 (m, 3H),4.08-4.09 (d, 1H), 3.92 (s, 1H), 3.12 (s, 3H), 2.93-3.00 (m, 2H), 2.37(s, 2H), 1.86-2.18 (m, 3H), 1.23-1.86 (m, 4H), 0.84-0.88 (t, 6H). LCMS:MH⁺1064, retention time 1.27 min.

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(83): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamoyl)-oxy)-methyl)-phenyl)-amino)-1-oxo-5-ureidopentan-2-yl)-amino)-3-methyl-1-oxobutan-2-yl)-carbamate(82) (360 mg, 0.34 mmol) in DMF (10 ml) was treated with piperidine(0.10 ml, 1.02 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (10-15%). The solvent was evaporatedunder vacuum to get 245 mg (86%) of4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(83) as an off-white solid. LCMS: MH⁺841, retention time 2.68 min.

4-((20S,23S)-1-azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(85):4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamate(83) (80 mg, 0.095 mmol), HATU (72.34 mg, 0.19 mmol), azido-peg5-acid(84) (38.28 mg, 0.114 mmol) and diisopropylamine (0.04 ml, 0.23 mmol)were mixed in DMF (5 ml) and stirred at ambient temperature undernitrogen atmosphere for 16 h. The reaction mixture was reduced todryness under vacuum and purified by column chromatography on a silicacartridge eluting with methanol/DCM gradient (1-2%). The solvent wasevaporated under vacuum and the material was finally purified by RPprep-HPLC to obtain two peaks that were separated for Example (85)(Peak-1: 10 mg, 9%) and (Peak-2: 6 mg, 6%) as an off-white solid. ¹H NMR(400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.12-8.14 (d, 1H), 8.04-8.06 (d,1H), 7.87-7.89 (d, 1H), 7.76-7.79 (d, 1H), 7.60-7.62 (d, 2H), 7.34-7.37(d, 2H), 7.31 (s, 1H), 5.44 (s, 2H), 5.40 (s, 2H), 5.28 (s, 3H), 5.07(s, 2H), 4.22-4.24 (m, 2H), 3.47-3.60 (m, 19H), 2.94-3.01 (m, 6H), 2.32(s, 3H), 2.18 (m, 2H), 1.23-1.89 (m, 12H), 0.81-0.89 (m, 9H). LCMS:MH⁺1158, retention time 2.51 min and 2.55 min.

Example 10: Synthesis of4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamate(87)

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamate(83) (245 mg, 0.29 mmol), HATU (221.55 mg, 0.58 mmol), azido-(PEG)₉-acid(86) (178.85 mg, 0.35 mmol) and diisopropylamine (0.12 ml, 0.73 mmol)were mixed in DMF (10 ml) and stirred at ambient temperature undernitrogen atmosphere for 6 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (1-2%). The solvent was evaporatedunder vacuum and the material was purified by RP prep-HPLC to obtain twopeaks that were separated to obtain Compound (87) (Peak-1: 108 mg, 28%)and (Peak-2: 126 mg, 32%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H),8.10-8.12 (d, 1H), 8.04-8.06 (d, 1H), 7.85-7.87 (d, 1H), 7.76-7.79 (d,1H), 7.60-7.62 (d, 2H), 7.35-7.37 (d, 2H), 7.31 (s, 1H), 6.51 (s, 1H),5.97 (s, 1H), 5.40-5.44 (d, 4H), 5.07 (s, 3H), 5.07 (s, 2H), 4.22-4.24(t, 1H), 3.47-3.60 (m, 36H), 2.94-3.01 (m, 4H), 2.44 (s, 1H), 2.35 (s,4H), 2.18 (m, 2H), 1.95-1.97 (m, 3H), 1.85-1.89 (m, 4H), 0.82-0.89 (m,9H). LCMS: MH⁺1334, retention time 2.53 min.

Example 11: Synthesis of4-((38S,41S)-1-azido-38-isopropyl-36,39-dioxo-41-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-37,40-diazadotetracontan-42-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(89)

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo-[de]-pyrano-[3′,4′:6,7]-indolizino-[1,2-b]-quinolin-1-yl)-carbamate(83) (80 mg, 0.09 mmol), HATU (72.34 mg, 0.19 mmol), azido-(PEG)ii-acid(88) (68.69 mg, 0.11 mmol) and diisopropylamine (0.04 ml, 0.23 mmol)were mixed in DMF (5 ml) and stirred at ambient temperature undernitrogen atmosphere for 16 h. The reaction mixture was reduced todryness under vacuum and purified by column chromatography on a silicacartridge eluting with methanol/DCM gradient (1-2%). The solvent wasevaporated under vacuum and the material was finally purified by RPprep-HPLC to obtain two peaks for that were separated for Example (89)(Peak-1: 8 mg, 6%) and (Peak-2: 6 mg, 5%), as an off-white solid. ¹H NMR(400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.10-8.12 (d, 1H), 8.04-8.06 (d,1H), 7.85-7.87 (d, 1H), 7.76-7.79 (d, 1H), 7.59-7.62 (d, 2H), 7.35-7.37(d, 2H), 7.31 (s, 1H), 5.44 (s, 2H), 5.40 (s, 2H), 5.29 (s, 3H), 5.07(s, 2H), 4.22-4.24 (m, 1H), 3.37-3.59 (m, 40H), 2.94-3.01 (m, 8H),2.32-2.66 (m, 8H), 1.79-1.97 (m, 10H), 0.81-0.89 (m, 9H). LCMS: MH⁺1422,retention time 2.53 min. Mass: (M+H) 1422

Example 12: Synthesis of methyl2-(4-((20S,23S)-1-azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(101)

2-Hydroxy-2-(4-nitrophenyl)acetonitrile (91): To a stirred solution of4-nitrobenzaldehyde (90) (5.0 g, 33.11 mmol) in DCM (60 mL) was addedTMSCN (4.26 g, 43.04 mmol) ZnI₂ (1.05 g, 3.31 mmol) at r.t. Theresultant reaction mixture was stirred at 60° C. for 3 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (15 mL) and extractedwith DCM (2×100 mL). The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to get crude compound.The crude compound was purified by flash chromatography eluting with 20%EtOAc in hexane to get 2.9 g (60% yield) of2-hydroxy-2-(4-nitrophenyl)acetonitrile (91) as a sticky liquid. ¹H NMR(400 MHz, DMSO-d₆): δ 8.30-8.32 (d, 2H), 7.76-7.78 (d, 2H), 7.40-7.42(d, 1H), 5.99-6.00 (d, 1H).

2-Hydroxy-2-(4-nitrophenyl)acetic acid (92): To a stirred solution of2-hydroxy-2-(4-nitrophenyl)acetonitrile (91) (13.0 g, 73.03 mmol) inAcOH (65 mL) was added 10(N) aqueous HCl (65 mL) at 10° C. The resultantreaction mixture was stirred at r.t for 6 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound 13 g (90% yield) of 2-hydroxy-2-(4-nitrophenyl) acetic acid(92) as a sticky solid. This crude compound was used for the next stepwithout further purification.

Methyl 2-hydroxy-2-(4-nitrophenyl)acetate (93): To a stirred solution of2-hydroxy-2-(4-nitrophenyl)acetic acid (92) (7 g, 35.53 mmol) in MeOH(70 ml), H₂SO₄ (5 ml) was added dropwise at 0° C. The resultant reactionmixture was stirred at 85° C. for 5 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was concentrated under reduced pressure and extracted with ethylacetate (2×50 ml) and water. The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound. The crude compound was purified by flash chromatographyeluting with 100% EtOAc to get 4.5 g (60% yield) of methyl2-hydroxy-2-(4-nitrophenyl) acetate (93) as a pale brown gum. ¹H NMR(400 MHz, DMSO-d₆): δ 8.21-8.24 (d, 2H), 7.68-7.70 (d, 2H), 6.45-6.46(d, 1H), 5.36-5.37 (d, 1H), 3.62 (s, 3H). LCMS: MH⁺210, retention time2.66 min.

Methyl 2-(4-aminophenyl)-2-hydroxyacetate (94): methyl2-hydroxy-2-(4-nitrophenyl)acetate (93) (4.5 g, 25.85 mmol) was taken inpar shaker vessel in presence of MeOH (80 ml). Then Pd-C (500 mg) wasadded to it and keep the reaction mixture at 40 psi under hydrogenatmosphere for 3h. The progress of the reaction was monitored by TLC.After completion of starting material, reaction mixture was filteredthrough sintered funnel. The filtrate was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 2% MeOH in DCM to get 2.5 g (59% yield) ofmethyl 2-(4-aminophenyl)-2-hydroxyacetate (94) as a pale brown gum. ¹HNMR (400 MHz, DMSO-d₆): δ 6.99-7.01 (d, 2H), 6.48-6.50 (d, 2H),5.67-5.68 (d, 1H), 5.07 (s, 2H), 4.89-4.90 (d, 1H), 3.57 (s, 3H).

Methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(95): methyl 2-(4-aminophenyl)-2-hydroxyacetate (94) (2.5 g, 13.81mmol), Fmoc-Cit-OH (76) (8.22 g, 20.71 mmol), EEDQ (10.23 g, 41.43 mmol)were mixed in DCM-THF (1:1) (100 ml) and stirred at ambient temperatureunder nitrogen for 16 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (5-10%). The solvent was evaporatedunder vacuum to obtain 2.5 g (33% yield) of methyl2-(4-((S)-2-((((9H-fluoren-9-yl) methoxy) carbonyl)amino)-5-ureidopentanamido) phenyl)-2-hydroxyacetate desired product(95) as an off-white solid. LCMS: MH⁺ 561, retention time 2.91 min.

Methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-chloroacetate(96): To a stirred solution of methyl 2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(95) (700 mg, 1.24 mmol) in THF (10 mL), SOCl2 (0.10 ml, 1.49 mmol) wasadded at 0° C. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 3% MeOH in DCM to get 0.4 g (55% yield) ofmethyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-chloroacetate(96) as an off-white solid. LCMS: MH⁺ 579, retention time 3.15 min.

Methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(97): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (300 mg, 0.76 mmol) in DMF (5 mL) was added CS₂CO₃ (498.17 mg, 1.52mmol) at 0° C. and stirred the reaction mixture for 15 min. Then methyl2-(4-((S)-2-((((9H-fluoren-9-yl) methoxy) carbonyl)amino)-5-ureidopentanamido) phenyl)-2-chloroacetate (96) (531.22 mg,0.91 mmol) and KI (380.71 mg, 0.91 mmol) were added. The resultantreaction mixture was stirred at r.t for 1 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was quenched with water (10 mL) and extracted with DCM(2×10 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to get crude compound The crudecompound was purified by flash chromatography eluting with 2.5% MeOH inDCM to get 0.11 g (38% yield) of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(97) as an off-white solid. LCMS: MH⁺934, retention time 2.93 min.

Methyl2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(98): A solution of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(97) (150 mg, 0.16 mmol) in DMF (5 ml) was treated with piperidine (0.04ml, 0.48 mmol) and the reaction mixture stirred at 0° C. under nitrogenatmosphere for 1 h. The progress of the reaction was monitored by TLC.After completion of starting material the reaction mixture was reducedto dryness under vacuum to get 0.11 g (96.2% yield) of methyl2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(98) crude as a sticky liquid. LCMS: MH⁺713, retention time 2.55 min.

Methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(99): To a stirred solution of Fmoc-L-Valine (79) (62.85 mg, 0.18 mmol)in DMF (5 mL) was added DIPEA (0.06 mL, 0.38 mmol), HATU (0.11 mg, 0.30mmol) and methyl2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(98) (110 mg, 0.15 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 3% MeOH inDCM to get 0.13 g (81% yield) of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(99) as a sticky solid. LCMS: MH⁺1034, retention time 3.07 min.

Methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(100): A solution of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate (99) (130 mg, 0.12mmol) in DMF (2 ml) was treated with piperidine (0.03 ml, 0.37 mmol) andthe reaction mixture stirred at 0° C. under nitrogen atmosphere for 1 h.The progress of the reaction was monitored by TLC. After completion ofstarting material the reaction mixture was reduced to dryness undervacuum to get 0.09 g (88% yield) of methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(100) crude as a sticky liquid. LCMS: MH⁺812, retention time 2.59 min.

Methyl2-(4-((20S,23S)-1-azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(101): To a stirred solution of Azido-PEG5-acid (84) (49.56 mg, 0.14mmol) in DMF (5 mL) was added DIPEA (0.05 mL, 0.30 mmol), HATU (93.66mg, 0.24 mmol) and methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(100) (100 mg, 0.12 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain two peaks for desired products(101-a) (6 mg, 5% yield) and (101-b) (2.5 mg, 2% yield) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆) Compound (101-a): δ 10.11 (s, 1H),8.14-8.16 (d, 1H), 8.08-8.10 (d, 1H), 7.867-7.88 (d, 1H), 7.66-7.68 (d,2H), 7.56-7.61 (m, 3H), 7.53 (s, 1H), 7.27 (s, 1H), 6.49 (s, 1H), 6.38(s, 1H), 5.99 (s, 1H), 5.42 (s, 4H), 5.29 (s, 2H), 4.20-4.36 (m, 2H),2.94-3.69 (m, 30H), 2.32-2.50 (m, 2H), 1.01-1.94 (m, 11H), 1.21-1.25 (t,5H), 0.81-0.88 (m, 9H). LCMS: MH⁺1129, retention time 2.42 and 2.43 min.

Example 13: Synthesis of Methyl2-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(102)

To a stirred solution of Azido-(PEG)₉-acid (75.61 mg, 0.14 mmol) in DMF(5 mL) was added DIPEA (0.05 mL, 0.30 mmol), HATU (93.66 mg, 0.24 mmol)and methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(12) (100 mg, 0.12 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to separate the desired products (102-a) (7 mg,5% yield) and (102-b) (14 mg, 9% yield) as an off-white solid. ¹H NMR(400 MHz, DMSO-d₆) of S-Isomer: δ 10.11 (s, 1H), 8.08-8.13 (m, 2H),7.84-7.86 (d, 1H), 7.66-7.68 (d, 2H), 7.53-7.61 (m, 4H), 7.27 (s, 1H),6.49 (s, 1H), 6.38 (s, 1H), 5.96 (s, 1H), 5.40-5.42 (d, 3H), 5.30 (s,2H), 4.36-4.38 (m, 1H), 4.20-4.24 (m, 1H), 3.69 (s, 3H), 3.29-3.60 (m,32H), 3.13-3.15 (d, 2H), 2.92-3.02 (m, 3H), 2.49-2.50 (d, 2H), 2.32-2.45(m, 1H), 1.84-1.87 (m, 1H), 1.36-1.40 (m, 2H), 1.21-1.25 (t, 3H),0.81-0.88 (m, 9H). LCMS: MH⁺1305, retention time 2.44 and 2.45 min.

Example 14: Synthesis of Methyl2-(4-((38S,41S)-1-azido-38-isopropyl-36,39-dioxo-41-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-37,40-diazadotetracontan-42-amido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(103)

To a stirred solution of Azido-(PEG)ii-acid (88) (88.93 mg, 0.14 mmol)in DMF (5 mL) was added DIPEA (0.05 mL, 0.30 mmol), HATU (93.66 mg, 0.24mmol) and methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(100) (100 mg, 0.12 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to separate the desired products (103-a) (14mg, 8% yield) and (103-b) (14 mg, 9% yield) as an off-white solid. ¹HNMR (400 MHz, DMSO-d₆) R isomer: δ 10.09 (s, 1H), 8.08-8.14 (m, 2H),7.84-7.87 (d, 1H), 7.66-7.68 (d, 2H), 7.53-7.60 (m, 4H), 7.27 (s, 1H),6.49 (s, 1H), 5.40-5.42 (d, 3H), 5.30 (s, 2H), 4.20-5.42 (m, 2H),3.29-3.69 (m, 40H), 2.93-3.16 (m, 6H), 2.32-2.38 (m, 3H), 1.84-1.87 (m,5H), 1.26-1.80 (m, 7H), 1.21-1.25 (t, 3H), 0.81-0.89 (m, 9H). LCMS:MH⁺1393, retention time of R isomer 2.44 min and S isomer 2.46 min.

Example 15: Synthesis of4-((20S,23S)-1-azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(108)

Tert-butyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(104): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (2 g, 5.102 mmol) in DMSO (6 mL) were added K₂CO₃ (7.052 g, 51.02mmol) and tert-butyl (3-bromopropyl) carbamate (14.579 g, 61.225 mmol)at r.t. The reaction mixture was stirred at room temperature. After 6 hthe reaction mixture was quenched with water and extracted by ethylacetate (2×15 mL). The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to get crude compound.The crude compound was purified by flash chromatography eluting with5-10% MeOH in DCM to get 1 g (33.1% yield) oftert-butyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(104) as an off-white solid. LCMS: MH⁺550, retention time 3.22 min.[00471](S)-9-(3-Aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105): A solution of tert-butyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(104) (1 g, 1.821 mmol) in Dioxane (30 mL) was treated with Dioxane-HCl(4M, 30 ml) at 0 c and the reaction mixture stirred at r.t undernitrogen atmosphere for 16h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol in DCM gradient (10-15%). The solvent wasevaporated under vacuum to obtain 0.6 g (61% yield) of(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) as a yellow solid. LCMS: MH⁺450, retention time 1.46 min.

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(106): To a stirred solution of(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) (0.6 g, 0.668 mmol) in DMSO (1 ml) was added(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(81) (1.3 g, 0.802 mmol) and TEA (516 mg, 2.004 mmol) The reaction wasstirred at ambient temperature under nitrogen atmosphere for 3h. Thereaction mixture was reduced to dryness under vacuum and purified bycolumn chromatography on a silica cartridge eluting with methanol/DCMgradient (5-10%). The solvent was evaporated under vacuum to obtaindesired product 0.5 g (45% yield) of4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamateFmoc product (106) was deprotected to the desired product (107) as abrown sticky solid, by reacting (106) with piperidine. LCMS: MH⁺855,retention time 2.71 min.

4-((20S,23S)-1-Azido-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosan-24-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(108):4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(107) (0.120 g, 0.141 mmol) in DMF (1 ml) were added Azido-(PEG)₅-acid(84) (56 mg, 0.169 mmol), diisopropyl ethylamine (54 mg, 0.422 mmol) andHATU (80 mg, 0.211 mmol). The reaction mixture was stirred at ambienttemperature under nitrogen atmosphere for 6h. The reaction mixture wasreduced to dryness under vacuum and purified by column chromatography ona silica cartridge eluting with methanol/DCM gradient (5-10%). Thesolvent was evaporated under vacuum and the material was finallypurified by RP prep-HPLC to obtain the desired product (108) (15 mg) asan off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.9 (s, 1H), 8.1 (d,1H), 8.06 (d, 1H), 7.86 (d, 1H), 7.59 (d, 2H), 7.49 (d, 2H), 7.28 (m,1H), 7.27 (m, 3H), 6.4 (s, 1H), 5.9 (m, 1H), 5.42 (m, 3H), 5.3 (s, 2H)4.9 (s, 2H), 4.3 (m, 1H), 4.2 (m, 3H), 3.5-2.9 (m, 26H), 1.97 (m, 3H),1.88 (m, 2H), 1.78 (m, 2H), 1.68 (d, 1H), 1.45 (m, 1H), 1.28 (t, 3H),0.95 (m, 9H). LCMS: MH⁺1172, retention time 2.48 min.

Example 16: Synthesis of4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(109)

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(107) (0.150 g, 0.176 mmol) in DMF were added Azido-(PEG)₉-acid (86) (98mg, 0.193 mmol) and diisopropyl ethylamine (67.94 mg, 0.527 mmol) andHATU (100.178 mg, 0.263 mmol).The reaction mixture was stirred atambient temperature under nitrogen atmosphere for 6h. The reactionmixture was reduced to dryness under vacuum and purified bychromatography on a silica cartridge eluting with methanol/DCM gradient(5-10%). The solvent was evaporated under vacuum and the material wasfinally purified by RP prep-HPLC to obtain the desired product (107) (16mg) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.9 (s, 1H), 8.1(d, 1H), 8.06 (d, 1H), 7.86 (d, 1H), 7.59 (d, 2H), 7.49 (d, 2H), 7.28(m, 1H), 7.27 (m, 3H), 6.4 (s, 1H), 5.9 (m, 1H), 5.42 (m, 3H), 5.3 (s,2H) 4.9 (s, 2H), 4.3 (m, 1H), 4.2 (m, 3H), 3.5-2.9 (m, 35H), 2.4 (m,2H), 1.97 (m, 3H), 1.88 (m, 2H), 1.78 (m, 2H), 1.58 (d, 1H), 1.45 (m,1H), 1.23 (t, 6H), 0.95 (m, 9H). LCMS: MH⁺1348, retention time 2.5 min.

Example 17: Synthesis of4-((38S,41S)-1-azido-38-isopropyl-36,39-dioxo-41-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-37,40-diazadotetracontan-42-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(110)

Scheme 17: Synthesis of protease-cleavable linker-payload conjugate(110).

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(107) (0.2 g, 0.234 mmol) in DMF (1 ml) were added Azido-peg11-acid (88)(168 mg, 0.281 mmol), diisopropyl ethylamine (90 mg, 0.703 mmol) andHATU (133.48 mg, 0.351 mmol).The reaction was stirred at ambienttemperature under nitrogen atmosphere for 6 h. The reaction mixture wasreduced to dryness under vacuum and purified by column chromatography ona silica cartridge eluting with methanol/DCM gradient (5-10%). Thesolvent was evaporated under vacuum and the material was finallypurified by RP prep-HPLC to obtain the desired product (110) (12 mg) asan off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.9 (s, 1H), 8.1 (d,1H), 8.06 (d, 1H), 7.86 (d, 1H), 7.59 (d, 2H), 7.49 (d, 2H), 7.28 (m,1H), 7.27 (m, 3H), 6.4 (s, 1H), 5.9 (m, 1H), 5.42 (m, 3H), 5.3 (s, 2H)4.9 (s, 2H), 4.3 (m, 1H), 4.2 (m, 4H), 3.5-2.9 (m, 51H), 2.4 (m, 2H),1.97 (m, 3H), 1.88-1.06 (m, 13H), 0.95 (m, 9H). LCMS: MH⁺1437, retentiontime 1.62 min.

Example 18: Synthesis of1-azido-N-((2S)-1-(((2S)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(122)

2-hydroxy-2-(4-nitrophenyl) acetic acid (112): To a stirred solution of1-(4-nitrophenyl) ethan-1-one (111) (5 g, 30.28 mmol), in1,4-dioxane/H₂O (40 mL, 3:1 by volume) was added SeO₂ (4 g, 36.34 mmol)followed by Yb(oTf)₃ (1.88 g, 3.03 mmol) under nitrogen atm at r.t. Theresultant reaction mixture was stirred at 110° C. for 16 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was cooled to 0° C., quenched with ice coldwater (100 mL) and extracted with EtOAc (2×200 mL). The combined organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to get 1.7 g (crude) of 2-hydroxy-2-(4-nitrophenyl) acetic acid(112) as a pale yellow semi solid. The crude compound was taken to nextstep without any further purification. ¹H NMR (400 MHz, CDCl₃): δ8.27-8.17 (m, 2H), 7.71-7.68 (m, 2H), 5.37 (s, 1H).

Methyl-2-hydroxy-2-(4-nitrophenyl)acetate (113): To a stirred solutionof 2-hydroxy-2-(4-nitrophenyl) acetic acid (112) (1.7 g, 8.62 mmol) inmethanol (20 mL) was added sulphuric acid (2 mL) at 0° C. The resultantreaction mixture was stirred at r.t for 3 h. The progress of thereaction was monitored by LCMS and TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by columnchromatography using silica gel (230-400 mesh) eluting with 30% EtOAc inpet ether to get 850 mg (47% yield) ofMethyl-2-hydroxy-2-(4-nitrophenyl)acetate (113) as an pale yellow solid.¹H NMR (400 MHz, CDCl₃): δ 8.23 (d, J=9.2 Hz, 2H), 7.66 (d, J=9.2 Hz,2H), 5.29 (s, 1H), 3.80 (s, 3H), 3.60 (d, J=5.1 Hz, 1H).

Methyl 2-(4-aminophenyl)-2-hydroxyacetate (114): To a stirred solutionof (Methyl 2-hydroxy-2-(4-nitrophenyl)acetate (113) (2.0 g, 9.47 mmol)in methanol (20 mL) was added Pd—C (200 mg) at r.t. The reaction mixturewas stirred at r.t under H₂ atm (balloon pressure) for 12 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was filtered through celite bedand the filtrate was concentrated under reduced pressure to get crudecompound. The crude compound was purified by column chromatography usingsilica gel (230-400 mesh) eluting 50% EtOAc in pet ether to get 1.3 g(76% yield) of methyl 2-(4-aminophenyl)-2-hydroxyacetate (114) as a paleyellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.0 (d, J=8.4 Hz, 2H), 6.50(d, J=8.4 Hz, 2H), 5.67 (d, J=5.2 Hz, 1H), 5.07 (s, 2H), 4.90 (d, J=5.2Hz, 1H), 3.57 (s, 3H).

Methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(115): To a stirred solution of methyl2-(4-aminophenyl)-2-hydroxyacetate (114) (1.0 g, 5.52 mmol) in THF/DCM(60 mL, 1:1 by volume) was added(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid(76) (2.63 g, 6.62 mmol) and EEDQ (4.10 g, 16.56 mmol) at r.t undernitrogen atmosphere The reaction mixture was stirred at r.t for 16 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with ice water andextracted with EtOAc (2×100 mL) to furnish crude compound. The crudecompound was triturated with diethyl ether (30 mL), filtered, driedunder vacuum to get 3.0 g (97% yield) of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(115) as an off-white solid. LC-MS: m/z 561.49 [(M+H)⁺)]; R_(t): 1.74min; 78.38% purity.

Methyl 2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-hydroxyacetate(116): To a stirred solution of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(115) (1.5 g, 2.68 mmol) in DMF (10 mL) was added piperidine (1.0 mL)under nitrogen atm at r.t. The resultant reaction mixture was stirred atr.t for 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound wastriturated with diethyl ether (30 mL) and ethyl acetate (30 mL),filtered, dried under vacuum to get 750 mg (83% yield) of methyl2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-hydroxyacetate (116) asan off-white solid. The crude compound was used in the next step withoutany further purification. LC-MS: m z 337.44 [(M−H)⁻-]; Rt: 1.68 min;57.36% purity.

Methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-ethylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(117): To a stirred solution of methyl2-(4-((S)-2-amino-5-ureidopentanamido)phenyl)-2-hydroxyacetate (116)(1.6 g, 4.73 mmol), in DMF (28 mL) was added HOAt (960 mg, 7.10 mmol),EDC.HCl (1.36 g, 7.10 mmol) and(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (1.28 g, 3.78 mmol)at 0° C. to r.t. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure and quenched with ice water (50 mL). The precipitated solid wasfiltered off and dried to get crude compound. The crude compound wastriturated with diethyl ether (30 mL), filtered, dried under vacuum toget 2.0 g (64% Yield) of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(117) as an off-white solid. LC-MS: m z 660.56 [(M+H)⁺]; R_(t): 1.79min; 69.96% purity.

Methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(118): To a stirred solution of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(117) (700 mg, 1.06 mmol) in DMF (10 mL) was added 30% piperidine in DMF(1.4 mL) under N₂ at r.t. The resultant reaction mixture was stirred atr.t for 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound wastriturated with diethyl ether (30 mL) and ethyl acetate (20 mL),filtered, dried under vacuum to get 350 mg (75% yield) of methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(118) as an off-white solid. LC-MS: m z 438.33 [(M+H)⁺)]; R_(t): 0.88min; 79.25% purity.

Methyl2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(120): To a stirred solution of methyl2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)phenyl)-2-hydroxyacetate(118) (200 mg, 0.46 mmol) in DMF (5 mL) was added DIPEA (0.12 mL, 0.69mmol), HATU (262 mg, 0.69 mmol) and1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) (174 mg, 0.46mmol) at 0° C. The resultant reaction mixture was stirred at r.t for 16h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was quenched with ice water andextracted with 10% MeOH/DCM (2×40 mL). The combined organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure toget a crude mass. The crude compound was triturated with diethyl ether(30 mL), filtered, dried under vacuum to get 180 mg (crude) of methyl2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(120) as an off-white solid. The crude compound was taken to next stepwithout any further purification. LC-MS: m z 799.57 [(M+H)⁺]; R_(t):1.41 min; 34.97% purity

2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (121): To a stirred solution of methyl2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(120) (180 mg, 0.23 mmol) in THF/H₂O (5.5 mL) was added LiOH·H₂O (19 mg,0.46 mmol) at 0° C., then the reaction mixture was stirred at r.t for 1h. The progress of the reaction was monitored by TLC and LCMS. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure and water was added followed by acidification with 1NHCl solution. The reaction mixture was extracted with 10% MeOH/DCM (2×50mL). The combined organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get a crude mass. The crudecompound was triturated with diethyl ether (30 mL) to get 130 mg (73%yield) of2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (121) as an off-white solid. LCMS: m/z 785.63 [(M+H)⁺)]; R_(t):1.31 min; 82.71% purity.

Ethyl 1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oate (125): To astirred solution of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (123)(1.5 g, 4.88 mmol) in THF (20 mL) was added sodium hydride (351 mg,14.64 mmol) at 0° C. The resultant reaction mixture was stirred at r.tfor 30 min. To the reaction mixture was added ethyl 3-bromopropanoate(124) (1.33 g, 7.32 mmol) and the reaction mixture was stirred at r.tfor 3 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quenched with icewater and extracted with EtOAc (2×100 mL). The combined organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressureto afford 1.2 g of (61% yield) of Ethyl1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oate (125) as a pale yellowsemi solid. ¹H NMR (400 MHz, CDCl₃): δ 4.22-4.13 (m, 2H), 3.75-3.51 (m,15H), 3.49-3.39 (m, 1H), 2.91 (t, J=6.8 Hz, 2H), 2.58-2.57 (m, 1H),1.31-1.17 (m, 9H), 0.88-0.81 (m, 3H).

1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119): To a stirredsolution of ethyl 1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oate (125)(1.2 g, 2.95 mmol) in THF/H₂O (12:1 by volume, 13 mL) was added LiOH·H₂O(371 mg, 8.85 mmol) at 0° C., then the reaction mass was stirred at r.tfor 6 h. The progress of the reaction was monitored by TLC and LCMS.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound wastriturated with diethyl ether and acidified with citric acid. The crudecompound was extracted with 10% MeOH/DCM (2×100 mL). The combinedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to get 540 mg (48% yield) of1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) as colourlessgum. ¹H NMR (400 MHz, DMSO-d₆): δ 3.58-3.49 (m, 20H), 3.47-3.33 (m, 4H),2.43-2.39 (m, 2H), 2.07 (m, 2H).

1-azido-N-((2S)-1-(((2S)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(122): To a stirred solution of2-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (121) (130 mg, 0.17 mmol) in DMF (1 mL) was added DIPEA (0.04 mL0.26 mmol), HOBt (34 mg, 0.26 mmol), EDC.HCl (38 mg, 0.26 mmol) and(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(16) (74 mg, 0.17 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water and extracted with 10% MeOH/DCM (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound waspurified by RP-Prep HPLC to afford 10.4 mg (5% yield) of1-azido-N-((2S)-1-(((2S)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(122) (HCl salt) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ9.95 (s, 1H), 8.68-8.60 (m, 1H), 8.10 (d, J=7.4 Hz, 1H), 7.87 (d, J=8.8Hz, 1H), 7.79 (d, J=10.8 Hz, 1H), 7.58 (t, J=8.4 Hz, 2H), 7.45-7.37 (m,2H), 7.32 (s, 1H), 6.26 (s, 2H), 5.48-5.26 (m, 7H), 5.00 (d, J=16.5 Hz,1H), 4.40 (q, J=5.5 Hz, 1H), 4.24 (t, J=7.4 Hz, 1H), 3.62-3.48 (m, 26H),3.16-2.96 (m, 5H), 2.39-2.32 (m, 5H), 2.10-1.85 (m, 5H), 1.70-1.23 (m,4H), 0.89-0.82 (m, 9H). LC-MS (method 1): m/z 1202.45 [(M+H)⁺]; R_(t):1.68, 1.69 min; 37.07+55.18% purity, HP-LC (method 1): R_(t): 3.79 min,3.83 min; 43.71+49.32% purity.

Example 19: Synthesis of1-azido-N-((2S)-1-(((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(119)

2-hydroxy-N,N-dimethyl-2-(4-nitrophenyl)acetamide (123): To a stirredsolution of 2-hydroxy-2-(4-nitrophenyl)acetic acid (92) (3.0 g, 15.22mmol) in THF (50 mL) was added DIPEA (2.65 mL, 15.22 mmol), HOBT (2.05g, 15.22 mmol), DCC (3.29 g, 15.99 mmol) and dimethylamine (1.23 g,15.22 mmol) at 0° C. The resultant reaction mixture was stirred at r.tfor 16 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was reduced todryness under vacuum and purified by column chromatography on a silicacartridge eluting with methanol/DCM gradient (1-2%). The solvent wasevaporated under vacuum to obtain 2 g (59% yield) of2-hydroxy-N,N-dimethyl-2-(4-nitrophenyl)acetamide (123) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20-8.23 (d, 2H), 7.62-7.64 (d,2H), 5.97-5.99 (d, 1H), 5.57-5.58 (d, 1H), 2.94 (s, 3H), 2.84 (s, 3H).

2-(4-aminophenyl)-2-hydroxy-N,N-dimethylacetamide (124):2-hydroxy-N,N-dimethyl-2-(4-nitrophenyl)acetamide (123) (2.5 g, 14.04mmol) was taken in par shaker vessel in presence of MeOH (80 ml). ThenPd—C (500 mg) was added to it and keep the reaction mixture at 40 psiunder hydrogen atmosphere for 3h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was filtered through sintered funnel. The filtrate wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 2% MeOH inDCM to get 1.8 g (66% yield) of2-(4-aminophenyl)-2-hydroxy-N,N-dimethylacetamide (124) as a stickyliquid. ¹H NMR (400 MHz, DMSO-d₆): δ 6.93-6.95 (d, 2H), 6.48-6.50 (d,2H), 5.10-5.12 (d, 1H), 4.93-4.95 (d, 1H), 2.77 (s, 6H), 1.69-1.73 (m,2H).

(9H-fluoren-9-yl)methyl((2S)-1-((4-(2-(dimethylamino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(125): 2-(4-aminophenyl)-2-hydroxy-N,N-dimethylacetamide (124) (1.3 g,6.70 mmol), Fmoc-Cit-OH (76) (3.99 g, 10.05 mmol), EEDQ (4.96 g, 20.10mmol) were mixed in DCM-THF (1:1) (100 ml) and stirred at ambienttemperature under nitrogen for 16 h. The reaction mixture was reduced todryness under vacuum and purified by column chromatography on a silicacartridge eluting with methanol/DCM gradient (5-10%). The solvent wasevaporated under vacuum to obtain 1.5 g (39% yield) of (9H-fluoren-9-yl)methyl((2S)-1-((4-(2-(dimethylamino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(125) as an off-white solid. LCMS: MH⁺574, retention time 2.94 min.

(9H-fluoren-9-yl)methyl((2S)-1-((4-(1-chloro-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(126): To a stirred solution of (9H-fluoren-9-yl) methyl((2S)-1-((4-(2-(dimethylamino)-1-hydroxy-2-oxoethyl) phenyl)amino)-1-oxo-5-ureidopentan-2-yl) carbamate (125) (1 g, 1.74 mmol) inTHF (15 mL), SOCl2 (0.24 ml, 2.09 mmol) was added at 0° C. The resultantreaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude compound was purified by flash chromatographyeluting with 3% MeOH in DCM to get 0.35 g (33% yield) of(9H-fluoren-9-yl)methyl((2S)-1-((4-(1-chloro-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(126) as an off-white solid. LCMS: MH⁺ 592, retention time 3.02 min.

(9H-fluoren-9-yl)methyl((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(127): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (120 mg, 0.30 mmol) in DMF (3 mL) was added CS₂CO₃ (149.45 mg, 0.45mmol) at 0° C. and stirred the reaction mixture for 15 min. Then(9H-fluoren-9-yl) methyl((2S)-1-((4-(1-chloro-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(126) (217.27 mg, 0.36 mmol) and KI (152.28 mg, 2.29 mmol) were added.The resultant reaction mixture was stirred at r.t for 1 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (10 mL) and extractedwith EtOAc (2×10 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound The crude compound was purified by flash chromatography elutingwith 2.5% MeOH in DCM to get 0.11 g (38% yield) of(9H-fluoren-9-yl)methyl((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(127) as an off-white solid. LCMS: MH⁺948, retention time 2.93 min.

(2S)-2-Amino-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(128): A solution of (9H-fluoren-9-yl)methyl((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(127) (80 mg, 0.08 mmol) in DMF (3 ml) was treated with piperidine(21.51 mg, 0.25 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.05 g (95.3% yield) of(2S)-2-amino-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(128) crude as a sticky liquid. LCMS: MH⁺726, retention time 2.37 min.

(9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(129): To a stirred solution of Fmoc-L-Valine (79) (34.22 mg, 0.10 mmol)in DMF (5 mL) was added DIPEA (0.03 mL, 0.21 mmol), HATU (63.91 mg, 0.16mmol) and(2S)-2-amino-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(128) (61 mg, 0.08 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 3% MeOH inDCM to get 0.05 g (57% yield) of (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-I-oxobutan-2-yl)carbamate(129) as a sticky solid. LCMS: MH⁺1047, retention time 2.89 min.

(2S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(130): A solution of (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-I-oxobutan-2-yl)carbamate(129) (115 mg, 0.11 mmol) in DMF (3 ml) was treated with piperidine(0.03 ml, 0.32 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.09 g of(2S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(130) crude as a sticky liquid and this material was used for the nextstep without further purification.

1-azido-N-((2S)-1-(((2S)-1-((4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(131): To a stirred solution of Azido-(PEG)₉-acid (86) (74.41 mg, 0.14mmol) in DMF (5 mL) was added DIPEA (0.05 mL, 0.30 mmol), HATU (92.18mg, 0.24 mmol) and(2S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(130) (100 mg, 0.12 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired products (131-S) and(131-R) (55 mg, 35% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): (10.09 (s, 1H), 8.12-8.14 (d, 1H), 8.07-8.09 (d, 1H),7.85-7.87 (d, 1H), 7.67-7.69 (d, 2H), 7.52-7.56 (m, 3H), 7.38 (s, 1H),7.27 (s, 1H), 6.49-6.52 (d, 2H), 5.97 (t, 1H), 5.41-5.42 (d, 4H), 5.31(s, 2H), 4.32-4.45 (m, 1H), 4.18-4.30 (t, 1H), 3.53-3.60 (m, 6H),3.48-3.50 (d, 24H), 3.37-3.44 (m, 2H), 3.26-3.32 (d, 4H), 3.14 (s, 4H),2.95-3.02 (m, 3H), 2.85 (s, 3H), 2.49-2.50 (d, 2H), 2.37-2.39 (m, 1H),1.95-1.97 (m, 1H), 1.84-1.88 (m, 3H), 1.32-1.78 (m, 4H), 1.25-1.28 (t,3H), 0.85-0.89 (m, 9H). LCMS: MH⁺1318, retention time 2.32 min.

Example 20: Synthesis of1-azido-N-((2S)-1-(((2S)-1-((4-(1-(((s)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(132)

To a stirred solution of Azido-(PEG)₉-acid (88) (87.52 mg, 0.14 mmol) inDMF (5 mL) was added DIPEA (0.05 mL, 0.30 mmol), HATU (92.18 mg, 0.24mmol) and(2S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(1-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-(dimethylamino)-2-oxoethyl)phenyl)-5-ureidopentanamide(130) (100 mg, 0.12 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired products (132-S) and(132-R) (12 mg, 7% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 10.09 (s, 1H), 8.12-8.14 (d, 1H), 8.07-8.09 (d, 1H),7.85-7.87 (d, 1H), 7.67-7.69 (d, 2H), 7.52-7.57 (m, 3H), 7.38 (s, 1H),7.27 (s, 1H), 6.49-6.52 (d, 2H), 5.96-5.99 (t, 1H), 5.41-5.42 (d, 4H),5.31 (s, 2H), 4.32-4.45 (m, 1H), 4.18-4.30 (t, 1H), 3.53-3.61 (m, 6H),3.48-3.51 (d, 33H), 3.38-3.44 (m, 2H), 3.32-3.37 (d, 1H), 3.26 (s, 1H),3.14 (s, 5H), 2.95-3.02 (m, 3H), 2.66 (s, 3H), 2.501-2.504 (d, 2H),2.39-2.46 (m, 1H), 1.95 (m, 1H), 1.84-1.88 (m, 2H), 1.32-1.78 (m, 4H),1.23-1.28 (m, 3H), 0.82-0.89 (m, 9H). LCMS: MH⁺1406, retention time 2.32min.

Example 21: Synthesis of1-Azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(141)

(4-Amino-2,6-difluorophenyl)methanol (134): To a stirred solution of(2,6-difluoro-4-nitrophenyl)methanol (133) (500 mg, 2.64 mmol) in MeOH(15 mL) was added Raney Ni (15.52 mg, 0.26 mmol) in presence of hydrogenballoon. The resultant reaction mixture was stirred at r.t for 35 min.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was filtered through sintered funneland the filtrate was concentrated under reduced pressure to get crudecompound 0.35 g (83% yield) of (4-amino-2,6-difluorophenyl)methanol(134) as a sticky solid. This crude compound was used for the next stepwithout further purification. ¹H NMR (400 MHz, DMSO-d₆) δ 6.12-6.15 (d,2H), 5.66 (s, 2H), 4.80-4.82 (t, 1H), 4.28-4.30 (d, 2H).

(9H-fluoren-9-yl)methyl(S)-(1-((3,5-difluoro-4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(135): (4-amino-2,6-difluorophenyl)methanol (134) (400 mg, 2.51 mmol),Fmoc-Cit-OH (76) (1198.49 mg, 3.01 mmol), EEDQ (1864.15 mg, 7.54 mmol)were mixed in DCM-THF (1:1) (50 ml) and stirred at ambient temperatureunder nitrogen for 16 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (5-10%). The solvent was evaporatedunder vacuum to obtain desired product 0.8 g (59% yield)(9H-fluoren-9-yl) methyl(S)-(1-((3,5-difluoro-4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(135) as a white solid. LCMS: MH⁺539, retention time 2.93 min.

(9H-fluoren-9-yl) methyl(S)-(1-((3,5-difluoro-4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(136): To a stirred solution of (9H-fluoren-9-yl) methyl(S)-(1-((3,5-difluoro-4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(135) (25 mg, 0.04 mmol) in ACN (10 mL), TMSCl (0.01 ml, 0.13 mmol) andsodium iodide (20.87 mg, 0.13 mmol) were added at OoC. The resultantreaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude compound was purified by flash chromatographyeluting with 1% MeOH in DCM to get 0.01 g (33% yield) of(9H-fluoren-9-yl)methyl(S)-(1-((3,5-difluoro-4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(136) as an off-white solid. LCMS: MH⁺649, retention time 3.47 min.

(9H-Fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(137): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (225 mg, 0.57 mmol) in DMF (5 mL) was added K₂CO₃ (118.90 mg, 0.86mmol) at 0° C. and stirred the reaction mixture for 15 min. Then(9H-fluoren-9-yl) methyl(S)-(1-((3,5-difluoro-4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(136) (557.69 mg, 0.86 mmol) was added. The resultant reaction mixturewas stirred at r.t for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wasquenched with water (10 mL) and extracted with DCM (2×10 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 2.5% MeOH in DCM to get0.22 g (42% yield) of (9H-fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(137) as an off-white solid.

(S)-2-Amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(138): A solution of (9H-fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(137) (250 mg, 0.27 mmol) in DMF (5 ml) was treated with piperidine(69.90 ml, 0.82 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.17 g (89% yield) of(S)-2-amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(138) crude as a sticky liquid. LCMS: MH⁺691, retention time 2.65 min.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(139): To a stirred solution of Fmoc-L-Valine (79) (112.03 mg, 0.33mmol) in DMF (5 mL) was added DIPEA (0.12 mL, 0.68 mmol), HATU (209.18mg, 0.55 mmol) and(S)-2-amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(138) (190 mg, 0.27 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for1 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with (3-6)% MeOHin DCM to get 0.21 g (75% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(139) as a sticky solid. LCMS: MH⁺1012, retention time 3.22 min.

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(140): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(139) (270 mg, 0.26 mmol) in DMF (2 ml) was treated with piperidine(0.07 ml, 0.80 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.201 g (95% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(140) crude as a sticky liquid and this material was used for the nextstep without further purification. LCMS: MH⁺790, retention time 2.71min.

1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(141): To a stirred solution of Azido-(PEG)₉-acid (86) (156.22 mg, 0.30mmol) in DMF (5 mL) was added DIPEA (0.11 mL, 0.63 mmol), HATU (193.52mg, 0.50 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(140) (201 mg, 0.25 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired product (141) (35 mg, 11%yield) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.44 (s,1H), 8.22-8.24 (d, 1H), 8.08-8.10 (d, 1H), 7.86-7.88 (d, 1H), 7.69 (s,1H), 7.51-7.53 (d, 1H), 7.43-7.46 (d, 2H), 7.27 (s, 1H), 6.50 (s, 1H),5.98 (s, 1H), 5.32-5.43 (m, 8H), 4.20-4.33 (m, 2H), 2.95-3.59 (m, 37H),2.32-2.56 (m, 5H), 1.42-1.97 (m, 7H), 1.30-1.34 (t, 3H), 0.87-0.89 (m,9H). LCMS: MH⁺1283, retention time 2.53 min.

Example 22: Synthesis of1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(142)

To a stirred solution of Azido-PEG1I-acid (88) (95.17 mg, 0.16 mmol) inDMF (5 mL) was added DIPEA (0.05 mL, 0.33 mmol), HATU (101.13 mg, 0.26mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(140) (105 mg, 0.13 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired product (142) (8 mg, 5%yield) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.44 (s,1H), 8.22-8.23 (d, 1H), 8.07-8.10 (d, 1H), 7.86-7.88 (d, 1H), 7.69 (s,1H), 7.51-7.54 (d, 1H), 7.43-7.45 (d, 2H), 7.27 (s, 1H), 6.50 (s, 1H),5.98 (s, 1H), 5.43 (s, 4H), 5.32 (s, 4H), 4.20-4.34 (m, 2H), 2.66-3.60(m, 37H), 2.32-2.50 (m, 6H), 1.33-1.97 (m, 7H), 1.30-1.33 (t, 3H),0.85-0.89 (m, 9H). LCMS: MH⁺1369, retention time 2.53 min.

Example 23: Synthesis of1-azido-N-((S)-1-(((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(147)

(9H-fluoren-9-yl)methyl((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(143): To a stirred solution Exatecan-Mesylate (16) (90 mg, 0.169 mmol)in DMF at 0° C. was added K₂CO₃ (110 mg, 0.339 mmol) and followed by(9H-fluoren-9-yl)methyl(S)-(1-((3,5-difluoro-4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(136) (130 mg, 0.203 mmol). The resultant reaction mixture was stirredat r.t for 1 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quenched with icewater and solid precipitate was filtered through sintered funnel and itwas dried under reduced pressure and purify under flash chromatographyand dried under vacuum to get 149 mg (91% yield) of(9H-fluoren-9-yl)methyl((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(143). LCMS: MH⁺956, retention time 3.47 min.

(S)-2-amino-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(144): To a stirred solution (9H-fluoren-9-yl)methyl((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(143) (149 mg, 0.156 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 10% MeOH in DCM to get 110 mg (96% yield) of(S)-2-amino-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(144). LCMS: MH 734, retention time 2.68 min.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(145): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (35 mg, 0.103 mmol),in DMF (5 mL) was added DIPEA (0.045 mL, 0.258 mmol), HATU (78 mg, 0.206mmol) and(S)-2-amino-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(144) (112 mg, 0.155 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum under low temperature. Then it was purify underflash chromatography and dried under vacuum to get 80 mg (73% yield) of(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(145). LCMS: MH⁺1055 retention time 3.60 min.

(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(146): To a stirred solution (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(145) (80 mg, 0.076 mmol) in DMF (3 ml), 30% piperidine in DMF (1 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 10% MeOH in DCM to get 60 mg (95% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(146). LCMS: MH 833, retention time 2.83 min.

1-azido-N-((S)-1-(((S)-1-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amid(147): To a stirred solution of Azido-(PEG)ii-acid (88) (30 mg, 0.05mmol), in DMF (5 mL) was added DIPEA (0.02 mL, 0.125 mmol), HATU (38 mg,0.1 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)-3,5-difluorophenyl)-5-ureidopentanamide(146) (62 mg, 0.075 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 4 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum to get crude compound. The crude compound waspurified by RP-prep-HPLC to get the desired compound (147), 5 mg (7%yield) isolated as a brown solid. ¹H NMR (400 MHz, DMSO-d₆): 10.30 (s,1H), 8.18 (d, 2H), 7.86 (d, 2H), 7.28-7.36 (t, 2H), 6.49 (d, 2H), 5.9(d, 2H), 5.41-5.32 (m, 5H), 5.19 (d, 1H), 4.33 (t, 1H), 4.23 (t, 2H),4.21 (t, 1H), 3.98-3.91 (m, 1H), 3.60-3.31 (m, 38H) 3.05-2.94 (m, 5H)2.38-2.26 (m, 5H), 2.14 (t, 2H), 1.98-1.89 (m, 5H), 1.87-1.81 (m, 2H),1.68 (m, 2H), 1.60 (m, 2H), 0.86 (s, 9H). LCMS: MH⁺1414, retention time3.02 min.

Example 24:1-azido-N-((S)-1-(((S)-1-((4-(2-(((1S,9s)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(148)

To a stirred solution of1-azido-N-((2S)-1-(((2S)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(122) (150 mg, 0.12 mmol) in DCM (2 mL) was added Dess- martinperiodinane (81 mg, 0.19 mmol) at 0° C. The resulting reaction mixturewas stirred at r.t for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wascooled to 0° C., quenched with ice cold water (10 mL) and extracted with10% MeOH in DCM (2×10 mL). The combined organic layer was washed withsaturated NaHCO₃ solution (20 mL). The separated organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to getcrude compound. The crude compound was purified by RP-preparative HPLCand the purified fractions was lyophilized to get 2.8 mg (2% yield) of1-azido-N-((S)-1-(((S)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(148) (HCl salt) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.46 (s,1H), 9.57 (d, J=8.6 Hz, 1H), 8.22 (d, J=7.0 Hz, 1H), 8.02 (d, J=8.7 Hz,2H), 7.87-7.80 (m, 4H), 7.32 (s, 1H), 6.52 (s, 1H), 6.00 (t, J=5.5 Hz,1H), 5.74 (q, J=6.3 Hz, 1H), 5.43-5.28 (m, 6H), 4.40 (t, J=6.3 Hz, 1H),4.25 (t, J=7.6 Hz, 1H), 3.61-3.48 (m, 26H), 3.20-2.90 (m, 4H), 2.39-2.30(m, 7H), 1.91-1.84 (m, 3H), 1.80-1.60 (m, 2H), 1.50-1.30 (m, 2H),0.89-0.83 (m, 9H). LC-MS (method 15): m/z 1200.77 [(M+H)⁺]; R_(t): 1.71min; 95.95% purity, HP-LC (method 15): R_(t): 4.87 min; 95.14% purity.

Example 25: Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(158)

(9H-fluoren-9-yl)methyl(S)-(6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate(150): Diisopropylethylamine (3.54 mL, 20.29 mmol), HATU (6.17 g, 16.23mmol) and (4-aminophenyl) methanol (75) (1 g, 8.12 mmol) were added to asolution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (6.0 g, 9.74 mmol) in DMF (30 mL) at room temperature, and thereaction mixture was stirred at room temperature for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced pressureand purified by combi-flash column chromatography, to afford(9H-fluoren-9-yl)methyl(S)-(6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate(150; 4 g) as a solid. LCMS: MH⁺730, retention time 2.49 min.

(S)-2-Amino-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(151): A solution of (9H-fluoren-9-yl)methyl(S)-(6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate(150), (2 g, 2.74 mmol) in DMF (20 ml) was treated with piperidine (0.81ml, 8.22 mmol) and the reaction mixture stirred at 0° C. under anatmosphere of nitrogen for 1 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, the reactionmixture was concentrated under reduced pressure and purified by flashchromatography eluting with 100% EtOAc to provide(S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(151; 1 g) as a gum. LCMS: MH⁺508, retention time 3.52 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(153): Diisopropylethylamine (2.57 mL, 14.77 mmol), HATU (4.49 g, 11.81mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (152)(2.74 g, 7.09 mmol) were added to a solution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(151) (3 g, 5.90 mmol) in DMF (25 mL) at 0° C., and the reaction mixturewas stirred at room temperature for 2 h. The progress of the reactionwas monitored by TLC. After completion of starting material, thereaction mixture was quenched with ice water, and the precipitated solidwas filtered and dried under vacuum, and then purified by flashchromatography to provide (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(153; 3 g) as a solid. LCMS: MH⁺877 retention time, 4.35 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(154): Pyridine (1.08 mL, 13.68 mmol) and 4-nitrophenyl chloroformate(2.06 g, 10.26 mmol) were added to a solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(153) (3 g, 3.42 mmol) in DCM (30 mL) at 0° C., and the reaction mixturewas stirred at room temperature for 2 h. The progress of the reactionwas monitored by TLC. After completion of starting material, thereaction mixture was concentrated under reduced pressure, and purifiedby flash chromatography eluting with 50% ethyl acetate in hexanes toafford (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(154; 3.4 g) as a gum. LCMS: MH⁺1042 retention time 2.78 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(155): Triethylamine (0.06 mL, 0.48 mmol) and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (130 mg, 0.24 mmol) were added to a solution(9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(154) (306 mg, 0.29 mmol) in DMSO (3.5 mL) at 0° C., and the reactionmixture was stirred at room temperature for 16h. The progress of thereaction was monitored by LCMS. After completion of starting material,the reaction mixture was quenched with water (15 mL) and theprecipitated out was filtered and passed through Combi-flash columnchromatography eluting with 5% MeOH in DCM to provide(9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(155; 0.220 g) as a solid. LCMS: MH⁺1339, retention time 2.68 min.

4-((S)-2-((S)-2-Amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(156): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(155), (150 mg, 0.11 mmol) in DMF (4 ml) was treated with piperidine(0.01 ml, 0.16 mmol) and the reaction mixture stirred at 0° C. under anitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure and purified by flash chromatographyeluting with 5% MeOH in DCM to afford4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(156; 130 mg) as a gum. LCMS: MH⁺1116, retention time 4.05 min.

4-((32S,35S)-1-Azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(157): Diisopropylethylamine (0.03 mL, 0.17 mmol), HATU (54.49 mg, 0.14mmol) and4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(156) (80 mg, 0.07 mmol) were added to a solution of azido-PEG9-acid(86, 47.43 mg, 0.08 mmol) in DMF (5 mL) at room temperature, and thereaction mixture was stirred at room temperature for 16 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced pressureand purified by combi-flash column chromatography to afford4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(157; 75 mg) as a solid. LCMS: MH⁺1609, retention time 2.20 min.

4-((32S,35S)-35-(4-Aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(158): A solution of trifluoroacetic acid (3 mL, 1% in DCM) was added toa solution of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(157) (90 mg, 0.05 mmol) in DCM (5 mL) at 0° C. and the reaction mixturewas stirred at room temperature for 1 h. The progress of the reactionwas monitored by LCMS. After completion of starting material, thereaction mixture was concentrated under reduced pressure and trituratedwith diethyl ether, then further purified by RP-prep-HPLC to obtainCompound (158) (Peak-1) (19 mg) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ10.04 (s, 1H), 8.23 (s, 1H), 8.06 (s, 2H), 7.76-7.79 (d, 1H),7.60-7.7.62 (d, 2H), 67.36-7.38 (d, 2H), 7.30 (s, 1H), 7.13-7.22 (m,5H), 5.44 (s, 2H), 5.36 (s, 2H), 5.18 (s, 2H), 4.56 (s, 2H), 4.37 (s,2H), 3.32-3.65 (m, 30H), 2.64-3.03 (m, 9H), 2.19-2.37 (m, 8H), 1.14-1.88(m, 11H), 0.85-0.88 (t, 3H), LCMS: MH⁺1353 retention time 2.40 and 2.44min.

Example 26: Synthesis of4-((35S,38S)-38-(4-aminobutyl)-1-azido-35-benzyl-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(169)

4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159): To a stirredsolution of (4-aminophenyl)methanol (75) (5.0 g, 40.61 mmol) in DMF (25mL) was added imidazole (5.54 g, 81.22 mmol) followed bytert-butyl(chloro)diphenylsilane (13.39 g, 48.73 mmol) at 0° C. Theresultant reaction mixture was stirred at r.t for 16 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (20 mL) and extractedwith EtOAc (2×200 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound The crude compound was purified by column chromatography usingsilica gel (230-400 mesh) eluting with 10% EtOAc in pet ether to get 6.6g (44% yield) of 4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159)as a gum. LCMS: m/z 362.31 [(M+H)⁺]; R_(t): 2.58 min; 93.68% purity.

(9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160): To a stirred solution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (5.0 g, 8.00 mmol) in DMF (50 mL) was added DIPEA (4.18 mL, 24.00mmol), HATU (6.08 g, 16.00 mmol) and4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159) (2.89 g, 8.00mmol) at 0° C. The resultant reaction mixture was stirred at r.t for 16h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was quenched with ice water. Theprecipitated solid was filtered off and dried under vacuum to get 5.5 g(71% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160) as a solid. LCMS: m/z 990.37 [(M+H)⁺]; R_(t): 2.84 min; 96.79%purity.

(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(ptolyl)methyl)amino)hexanamide (161): To a stirred solution of(9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160) (5.5 g, 5.68 mmol) in DMF (38.5 mL) was added piperidine (16.5 mL)at r.t. The resultant reaction mixture was stirred at r.t for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified bycolumn chromatography using silica gel (230-400 mesh) eluting with 100%EtOAc to get 3.5 g (83% yield) of(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(160) as a gum. LCMS: m/z 744.24 [(M−H)-]; R_(t): 2.20 min; 90.16%purity.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(162): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (152) (1.82 g, 4.69mmol), in DMF (35 mL) was added DIPEA (2.45 mL, 14.07 mmol), HATU (3.57g, 9.38 mmol) and(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(161) (3.5 g, 4.69 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 3.9 g (75% yield) of((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(162) as a solid. LCMS: m/z 1137.66 [(M+H)⁺]; R_(t): 2.96 min; 88.50%purity.

(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(163): To a stirred solution of(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(162) (1.0 g, 0.90 mmol) in DMF (7 mL) was added piperidine (3 mL) atr.t. The resultant reaction mixture was stirred at r.t for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified bycolumn chromatography using silica gel (230-400 mesh) eluting with 100%EtOAc to get 0.62 g (77% yield) of(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(163) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.08 (s, 1H), 8.08 (d,J=7.8 Hz, 1H), 8.07-7.63 (m, 4H), 7.57-7.55 (m, 2H), 7.49-7.37 (m, 11H),7.28-7.23 (m, 9H), 7.19-7.18 (m, 4H), 7.15-7.04 (m, 6H), 4.72 (s, 1H),4.45 (d, J=6.4 Hz, 1H), 3.46 (q, J=4.2 Hz, 1H), 2.96-2.92 (m, 1H),2.67-2.61 (m, 1H), 2.49-2.41 (m, 1H), 2.21 (s, 3H), 1.99-1.91 (m, 3H),1.71-1.41 (m, 6H), 1.03 (s, 9H).

1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(165): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oic acid(164) (311 mg, 0.56 mmol) in DMF (5 mL) was added DIPEA (0.3 mL, 1.68mmol), HATU (319 mg, 0.84 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(163) (500 mg, 0.56 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (20 mL) and extracted with EtOAc (2×200 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound waspurified by column chromatography using silica gel (230-400 mesh)eluting with 5% MeOH in DCM to get 0.51 g (64% yield) of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(165) as a gum. LCMS: m z 1431.51 [(M+H)⁺]; R_(t): 2.58 min; 85.28%purity.

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(166): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(165) (500 mg, 0.35 mmol) in methanol (5 mL) was added NH₄F (129 mg,3.49 mmol) at r.t. The resultant reaction mixture was stirred at r.t for6 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get a crude residue. The residue obtained was diluted withwater (20 mL) and extracted with EtOAc (2×200 mL). The combined organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to get crude compound. The crude compound was purified bycolumn chromatography using silica gel (230-400 mesh) eluting with 5%MeOH in DCM to get 0.39 g (94% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(166) as a gum. LCMS: m/z 1193.09 [(M+H)⁺]; R_(t): 1.87 min; 65.88%purity.

4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl(4-nitrophenyl) carbonate (167): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-amide(166) (390 mg, 0.33 mmol) in DCM (4 mL) was added pyridine (0.13 mL,1.64 mmol), 4-nitrophenyl chloroformate (14) (132 mg, 0.65 mmol) at 0°C. The resultant reaction mixture was stirred at r.t for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified bycolumn chromatography using silica gel (230-400 mesh) eluting with 5%MeOH in DCM to get 0.145 g (33% yield) of4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl(4-nitrophenyl) carbonate (167) as a gum. LCMS: m/z 1357.77 [(M+H)⁺];R_(t): 2.21 min; 81.14% purity.

4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(168): To a stirred solution4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl(4-nitrophenyl) carbonate (167) (140 mg, 0.10 mmol) in DMF (1.12 mL) wasadded pyridine (0.28 mL), HOBt (14 mg, 0.10 mmol) and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (11) (55 mg, 0.10 mmol) at 0° C. The resultant reactionmixture was stirred at r.t for 16 h. The progress of the reaction wasmonitored by LCMS. After completion of starting material, reactionmixture was quenched with water (20 mL) and extracted with EtOAc (2×200mL). The combined organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get crude compound. The crudecompound was purified by trituration with diethyl ether, filtered anddried under vacuum to get 0.13 g (76% yield) of4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(168) as a solid. LCMS: m/z 1653.85 [(M+H)⁺]; R_(t): 2.16 min; 60.47%purity.

4-((35S,38S)-38-(4-aminobutyl)-1-azido-35-benzyl-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(169): To a stirred solution of 1% TFA, 10% triisopropylsilane in DCM (4mL) was added4-((35S,38S)-1-azido-35-benzyl-38-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(168) (130 mg, 0.08 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 1 h. The progress of the reaction was monitored byLCMS. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by RP-prep HPLC to furnish 17 mg (16% yield) of4-((35S,38S)-38-(4-aminobutyl)-1-azido-35-benzyl-33,36-dioxo-3,6,9,12,15,18,21,24,27,30-decaoxa-34,37-diazanonatriacontan-39-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(169) (TFA salt) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.02 (s, 1H),8.25 (d, J=7.9 Hz, 1H), 8.07 (d, J=7.8 Hz, 2H), 7.79 (d, J=10.9 Hz, 1H),7.62 (d, J=8.1 Hz, 5H), 7.38 (d, J=8.3 Hz, 2H), 7.32 (s, 1H), 7.22 (m,4H), 6.52 (s, 1H), 5.44 (s, 2H), 5.29 (s, 3H), 5.09 (s, 2H), 4.56 (m,J=4.3 Hz, 1H), 4.40 (q, J=7.2 Hz, 1H), 3.49 (d, J=7.0 Hz, 42H), 3.12 (d,J=16.9 Hz, 2H), 3.02 (q, J=6.0 Hz, 1H), 2.77 (q, J=7.7 Hz, 3H), 2.38 (s,3H), 2.31 (t, J=6.0 Hz, 2H), 2.15-2.12 (m, 2H), 1.88-1.86 (m, 2H),1.65-1.62 (m, 4H), 1.33 (t, J=8.1 Hz, 2H), 0.87 (t, J=7.3 Hz, 3H). LC-MS(method 2): m/z 1397.30 [(M+H)⁺]; R_(t): 1.77 min; 98.23% purity, HP-LC(method 2): R_(t): 4.21 min; 95.13% purity.

Example 27: Synthesis of4-((32s,35s)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-2,6-difluorobenzyl((1s,9s)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(170).

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2,6-difluorobenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(146) (90 mg, 0.10 mmol), HATU (78.04 mg, 0.20 mmol), azido-peg9-acid(88) (67.93 mg, 0.12 mmol) and DIPEA (0.04 ml, 0.25 mmol) were mixed inDMF (5 ml) and stirred at ambient temperature under nitrogen atmospherefor 16 h. The reaction mixture was reduced to dryness under vacuum andpurified by column chromatography on a silica cartridge eluting withmethanol/DCM gradient (1-2%). The solvent was evaporated under vacuumand the material was finally purified by RP prep-HPLC to obtain twopeaks for products (170) (Peak-1) (17 mg, 12%) and as the undesiredisomer (Peak-2) (22 mg, 16%) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 10.40 (s, 1H), 8.21-8.22 (d, 1H), 8.03-8.05 (d, 1H),7.86-7.88 (d, 1H), 7.75-7.78 (d, 1H), 7.36-7.39 (d, 2H), 7.30 (s, 1H),6.50 (s, 1H), 5.99 (s, 1H), 5.43 (s, 4H), 5.26 (s, 3H), 5.08-5.11 (m,2H), 4.32 (s, 1H), 4.21 (s, 1H), 3.11-3.59 (m, 36H), 2.16-2.50 (m, 6H),1.84-1.96 (m, 7H), 0.81-0.89 (m, 9H). LCMS: MH⁺1370, retention time 2.57min.

Example 28: Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(174)

(9H₁-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(171): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(154) (417.33 mg, 0.40 mmol) in DMSO (5 mL) was added TEA (0.09 mL, 0.66mmol), and(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (105) (150 mg, 0.33 mmol) at 0° C. Theresultant reaction mixture was stirred at r.t for 4h. The progress ofthe reaction was monitored by LCMS. After completion of startingmaterial, reaction mixture was quenched with water (15 mL) and solidprecipitated out. It was filtered and the solid material was passedthrough Combi-flash column chromatography eluting with 5% MeOH in DCM toget 0.15 g (33% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′: 6,7] indolizino [1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(171) as an off-white solid. LCMS: MH⁺1353. Retention time, 4.76 min.

4-((S)-2-((S)-2-Amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(172): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(171) (150 mg, 0.11 mmol) in DMF (4 ml) was treated with piperidine(0.03 ml, 0.33 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 5% MeOH inDCM to get 0.10 mg (80% yield) of4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(172) as a pale brown gum, This material was used for the next step.

4-((32S,35S)-1-Azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(173): To a stirred solution of Azido-PEG9-Acid (86) (58.55 mg, 0.10mmol) in DMF (5 mL) was added DIPEA (0.03 mL, 0.22 mmol), HATU (67.27mg, 0.17 mmol) and4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(172) (100 mg, 0.08 mmol) at r.t. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure and purify by combi-flash columnchromatography, to get 65 mg (45% yield) of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(173) as an off-white solid.

4-((32S,35S)-35-(4-Aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(174): To a stirred solution of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(173) (65 mg, 0.04 mmol) in DCM 3 ml 1% TFA in DCM was added at 0° C.and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finaly purified by RP-prep-HPLC to obtain the desiredproduct (174) (15 mg, 27% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 10.05 (s, 1H), 8.24 (s, 1H), 8.06-8.09 (d, 2H), 7.58-7.60(d, 2H), 7.48-7.51 (d, 2H), 7.36 (s, 1H), 7.13-7.30 (m, 8H), 6.51 (s,1H), 5.42 (s, 2H), 5.30 (s, 2H), 4.96 (s, 2H) 4.56 (s, 1H), 4.36 (s,1H), 4.22 (s, 2H), 3.00-3.60 (m, 39H), 2.30-2.78 (m, 5H), 1.37-1.96 (m,12H), 1.28-1.32 (t, 3H), 0.85-0.89 (t, 3H).LCMS: MH⁺1367 retention time2.36.

Example 29: Synthesis ofN-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182-S) &N-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182)

Methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(175): To a stirred solution of methyl2-(4-aminophenyl)-2-hydroxyacetate (114) (600 mg, 3.31 mmol) in DMF (10mL) was addedN²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(diphenyl(p-tolyl)methyl)-L-lysine(149) (2.27 g, 3.64 mmol), HATU (1.89 g, 4.97 mmol) and DIPEA (1.73 mL,9.93 mmol) at r.t under nitrogen atmosphere The reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water (50 mL) and the precipitated solid was filteredoff, dried under vacuum to get crude compound. The crude compound wastriturated with pet ether (30 mL) and filtered off, dried under vacuumto get 2.5 g (96% yield) of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(175) as an off-white solid. LC-MS: m z 810.44 [(M+Na)⁺]; R_(t): 2.16min; 88.79% purity.

methyl2-(4-((S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(176): To a stirred solution of methyl2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(175) (2.5 g, 3.17 mmol) in acetonitrile (30 mL) was added piperidine(2.5 mL) under nitrogen atm at r.t. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 3-5% MeOH in DCM to get 1.7 g (95% yield) of methyl2-(4-((S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(176) as a pale yellow solid. LC-MS: m/z 564.43 [(M−H)−]; R_(t): 1.44min; 89.15% purity.

2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(177): To a stirred solution of methyl2-(4-((S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(176) (1.7 g, 3.01 mmol) in DMF (20 mL) was added(((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (152) (1.28 g, 3.31mmol), HATU (1.72 g, 4.52 mmol) and DIPEA (1.58 mL, 9.03 mmol) at r.t.The resultant reaction mixture was stirred at r.t for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with ice water (50 mL) and theprecipitated solid was filtered off, dried under vacuum to get crudecompound. The crude compound was purified by column chromatography usingsilica gel (100-200 mesh) eluting with 35-40% ethyl acetate in pet etherto get 2.25 g (80% Yield) of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(177) as an off-white solid. LC-MS: m z 957.23 [(M+Na)⁺]; R_(t): 2.18min; 84% purity.

Methyl2-(4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(178): To a stirred solution of methyl2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(177) (2.25 g, 2.41 mmol) in acetonitrile (25 mL) was added piperidine(2.25 mL) under nitrogen atm at r.t. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 3-5% MeOH in DCM to get 1.3 g (76% yield) of methyl2-(4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(178) as an off-white solid. LC-MS: m z 713.25 [(M+H)⁺]; R_(t): 1.43min; 84.110% purity.

Methyl2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(179): To a stirred solution of methyl2-(4-((S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)phenyl)-2-hydroxyacetate(178) (1.3 g, 1.82 mmol) in DMF (10 mL) was added1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) (763 mg, 2.01mmol), PyBOP (1.42 g, 2.73 mmol) and DIPEA (0.95 mL, 5.46 mmol) at r.t.The resultant reaction mixture was stirred at r.t for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with ice water (50 mL) and theprecipitated solid was filtered off, dried under vacuum to get crudecompound. The crude compound was purified by column chromatography usingsilica gel (100-200 mesh) eluting with 3-5% MeOH in DCM to get 1.4 g(71% yield) of methyl2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(179) as a pale brown sticky liquid. LC-MS: m z 1074.28 [(M+H)⁺]; R_(t):1.88 min; 88.77% purity.

2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (180): To a stirred solution of methyl2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyacetate(179) (1.4 g, 1.30 mmol) in THF-H₂O (33 mL, 10:1 by volume) was addedLiOH·H₂O (218 mg, 5.20 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get a crude mass. To the crudecompound water (20 mL) was added followed by acidification withsaturated citric acid solution. The precipitated solid was filtered off,dried under vacuum to get crude compound. The crude compound wastriturated with diethyl ether (20 mL) and filtered off, dried undervacuum to get 1.2 g (87% yield) of2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (180) as a pale yellow solid. LC-MS: m z 1060.63 [(M+H)⁺]; R_(t):1.90 min; 85.53% purity.

1-azido-N-((2S)-1-(((2S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(181): To a stirred solution of2-(4-((23S,26S)-1-azido-23-benzyl-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-2-hydroxyaceticacid (180) (50 mg, 0.05 mmol) in DMF (1 mL) was added(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (27 mg, 0.05 mmol), HATU (30 mg, 0.08 mmol) andDIPEA (0.03 mL 0.15 mmol) at r.t. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water (10 mL) and the precipitated solid was filteredoff, dried under vacuum to get crude compound. The crude compound wastriturated with diethyl ether (30 mL) and filtered off, dried undervacuum to get 350 mg (84% yield) of1-azido-N-((2S)-1-(((2S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(181) as a pale brown solid. LC-MS: m z 1478.90, 1478.15 [(M+H)⁺];R_(t): 2.10, 2.13 min; 32.06+42.40% purity (mixture of diastereomers).

N-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182-S) &N-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182-R): To a stirred solution of1-azido-N-((2S)-1-(((2S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(12) (350 mg, 0.24 mmol) in DCM (5 mL) was added a mixture of 1% TFA and10% TIPS in DCM (15 mL) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by RP-preparative HPLC to afford 17.4 mg (6%yield) ofN-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182-S) (HCl salt) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.02(s, 1H), 8.70 (d, J=8.8 Hz, 1H), 8.25 (d, J=7.6 Hz, 1H), 8.09 (d, J=8.0Hz, 1H), 7.80 (d, J=11.2 Hz, 1H), 7.69-7.59 (m, 5H), 7.46 (d, J=8.4 Hz,2H), 7.32 (s, 1H), 7.26-6.99 (m, 5H), 6.53 (s, 1H), 6.15 (s, 1H),5.52-5.47 (m, 1H), 5.43 (s, 2H), 5.33-5.28 (m, 1H), 5.21-5.16 (m, 1H),5.00 (s, 1H), 4.59-4.54 (m, 1H), 4.44-4.39 (m, 1H), 3.60-3.38 (m, 25H),3.16-3.13 (m, 2H), 3.06-3.01 (m, 1H), 2.78 (q, J=8.8 Hz, 3H), 2.39 (s,3H), 2.32 (t, J=4.4 Hz, 2H), 2.13-2.11 (m, 2H), 1.91-1.83 (m, 2H),1.81-1.75 (m, 1H), 1.69-1.52 (m, 3H), 1.41-1.35 (m, 2H), 0.88 (t, J=7.2Hz, 3H). LC-MS: m z 1221.77 [(M+H)⁺]; R_(t): 1.61 min; 98.06% purity,HP-LC: R_(t): 12.00 min; 99.39% purity. (182-R) Peak-B was 4.6 mg (2%yield) ofN-((2S)-1-(((2S)-6-amino-1-((4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-amide(182-R) (HCl salt) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.06(s, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.28 (d, J=7.2 Hz, 1H), 8.11 (d, J=7.6Hz, 1H), 7.78 (d, J=9.5 Hz, 4H), 7.59 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.0Hz, 2H), 7.32-7.05 (m, 6H), 6.54-6.25 (m, 2H), 5.55-5.43 (m, 3H), 5.23(q, J=15 Hz, 2H), 5.04 (s, 1H), 4.61-4.51 (m, 1H), 4.45-4.35 (m, 1H),3.59-3.49 (m, 25H), 3.15-3.03 (m, 3H), 2.85-2.72 (m, 3H), 2.38-2.28 (m,5H), 2.16-2.07 (m, 2H), 1.89-1.73 (m, 3H), 1.66-1.54 (m, 3H), 1.38-1.31(m, 2H), 0.87 (t, J=6.8 Hz, 3H). LC-MS (method 28): m/z 1221.77[(M+H)⁺]; R_(t): 1.63 min; 95.03% purity, HP-LC (method 28): R_(t):12.17 min; 95.72% purity.

Example 30: Synthesis of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-henzn[de]pyrann[3′,4′:6,7]indolizino[1,2-h]quinolin-1-yl)carbamate(186)

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide(183):(9H-fluoren-9-yl)methyl-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (80) (720 mg, 0.34 mmol) inDMF (5 ml) was treated with piperidine (0.20 ml, 1.02 mmol) and thereaction mixture stirred at 0° C. under nitrogen atmosphere for 1 h. Thereaction mixture was reduced to dryness under vacuum and purified bycolumn chromatography on a silica cartridge eluting with methanol/DCMgradient (10-15%). The solvent was evaporated under vacuum to obtain 490mg (86% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (183) as an off-white solid. LCMS: MH⁺380,retention time 1.22 min.

7-Azido-N-((S)-1-(((S)-1-((4-(hydroxymethyl) phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)heptanamide(184):(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide(183) (450 mg, 1.187 mmol), HATU (902 mg, 2.375 mmol), 7 azido heptanoicacid (264 mg, 1.544 mmol) and N-ethyldiisopropylamine (0.419 ml, 2.375mmol) were mixed in DMF (5 ml) and stirred at ambient temperature undernitrogen atmosphere for 6 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (1-2%). The solvent was evaporatedunder vacuum and the material was finally purified by columnchromatography on a silica cartridge eluting with methanol/DCM gradient(5-10%). The solvent was evaporated under vacuum to obtain 400 mg(63.25% yield) of 7-azido-N-((S)-1-(((S)-1-((4-(hydroxymethyl) phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)heptanamide (184) as a sticky liquid. ¹HNMR (400 MHz, DMSO-d₆) δ 9.91 (s, 1H), 8.3 (d, 1H), 8.21 (d, 1H), 8.07(d, 1H), 7.85 (d, 1H), 7.55 (d, 2H), 7.23 (d, 2H), 5.9 (t, 1H), 5.76 (s,2H), 5.4 (m, 1H), 4.3 (m, 1H), 4.2 (in, 1H), 3.3 (m, 3H), 2.99 (in, 2H),2.17 (m, 2H), 2.13 (m, 1H),1.58 (m, 1H), 1.50 (m, 1H), 1.48 (m, 5H),1.36-1.18 (m, 8H), 0.86-0.825 (m, 6H), LCMS: MH⁺533.5, retention time1.59 min.

4-((S)-2-((S)-2-(7-Azidoheptanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (185):7-azido-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)heptanamide(184) (250 mg, 0.47 mmol), 4-nitrophenyl chloroformate (25) (283 mg,1.41 mmol) were treated with pyridine (148 mg, 1.88 mmol) in DMF/THF (4ml) solvent and the reaction mixture was stirred at ambient temperatureunder nitrogen atmosphere for 4 h. The reaction mixture was reduced todryness under vacuum and purified by column chromatography on a silicacartridge eluting with methanol/DCM gradient (2%). The solvent wasevaporated under vacuum to obtain 180 mg (54% yield) of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (185) as a white solid. LCMS: MH⁺697,retention time 1.83 min.

4-((S)-2-((S)-2-(7-Azidoheptanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(186): 4-((S)-2-((S)-2-(7-azidoheptanamido)-3-methylbutanamido)-5ureidopentanamido) benzyl (4-nitrophenyl) carbonate (185) (80 mg, 0.118mmol), Exatecan mesylate (16) (51 mg, 0.118 mmol) and TEA (37 mg, 0.353mmol) were mixed in DMSO (0.5 ml) and stirred the reaction mixture atambient temperature under nitrogen for 2h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was concentrated under reduced pressure to get crude compound.The crude compound was purified by RP-prep HPLC to obtain the desiredproduct (186) as an off-white solid (21 mg, 17%). ¹H NMR (400 MHz,DMSO-d₆) δ 9.98 (s, 1H), 8.065 (d, 2H), 7.8 (m, 2H), 7.61 (d, 2H), 7.37(d, 2H), 7.31 (s, 1H),6.51 (s, 1H), 5.9 (q, 1H), 5.44-5.40 (d, 4H), 5.29(s, 2.H), 5.07 (s, 2H), 4.38 (d, 1H), 4.2 (t, 1H), 3.3-3.1 (m, 6H), 2.14(m, 4H),1.98-1.95 (m, 6H), 1.48 (m, 4H), 1.27 (m, 4H), 0.858 (m, 9H).LCMS: MH⁺994, retention time 2.64 min.

Example 31: Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(193)

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187): To a stirred solution of(((N-(9-Fluorenylmethoxycarbonyl)-L-valine (79) (1 g, 2.94 mmol), in DMF(20 mL) was added DIPEA (1.54 mL, 8.83 mmol), HATU (2.24 g, 5.89 mmol)and(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(161) (2.19 g, 2.94 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 2.5 g (79% yield) of (((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187) as a solid. LCMS: MH⁺1067, retention time 2.42 min.

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(188): To a stirred solution of (((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187) (1.5 g, 1.40 mmol) in DMF (6 mL), 30% piperidine in DMF (4.5 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for2 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 1.1 g (97% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(188) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.07 (s, 1H), 7.64-7.63(d, 4H), 7.56-7.54 (d, 2H), 7.46-7.35 (m, 9H), 7.27-7.24 (m, 8H),7.185-7.11 (m, 2H), 7.05-7.03 (d, 2H), 4.71 (s, 2H), 4.44 (d, 1H),3.25-3.16 (d, 1H), 3.01-3.00 (m, 1H), 2.21 (s, 3H), 1.98-1.93 (m, 2H),1.68-1.38 (m, 4H), 1.15 (s, 10H), LCMS: MH⁺845, retention time 3.63 min.

1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (484mg, 0.94 mmol) in DMF (8 mL) was added DIPEA (0.49 mL, 2.83 mmol), HATU(719.47 mg, 1.89 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(188) (800 mg, 0.94 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM to get 0.60g (51% yield) of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189) as a gum. ¹H NMR (400 MHz, DMSO-d₆): δ 9.91 (s, 1H), 8.02-8.00 (d,2H), 7.95 (s, 2H), 7.87-7.85 (d, 1H), 7.64-7.63 (d, 4H), 7.57-7.55 (d,2H), 7.46-7.32 (m, 11H), 7.26-7.24 (m, 8H), 7.15-7.11 (t, 2H), 7.05-7.03(d, 2H), 4.71 (s, 2H), 4.35-4.33 (m, 1H), 4.19 (s, 1H), 3.59-3.36 (m,38H), 2.68-2.38 (m, 6H), 2.22 (s, 3H), 1.98-1.92 (m, 2H), 1.47-1.17 (m,4H), 1.02 (s, 9H), 0.85-0.80 (m, 6H). LCMS: MH⁺1338, retention time 2.92min.

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189) (600 mg, 0.44 mmol) in methanol (10 mL) was added NH₄F (166 mg,4.48 mmol) at r.t. The resultant reaction mixture was stirred at r.t for6 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get a crude residue. The residue obtained was diluted withwater (15 mL) and extracted with EtOAc (2×20 mL). The combined organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 5% MeOH in DCM to get 0.40 g (81% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190) as a gum. ¹H NMR (400 MHz, DMSO-d₆): δ 9.81 (s, 1H), 7.96-7.94 (d,1H), 7.84-7.81 (d, 1H), 7.53-7.51 (d, 2H), 7.37-7.35 (d, 4H), 7.26-7.12(m, 9H), 7.096-7.04 (d, 2H), 5.06-5.04 (t, 1H), 4.43-4.41 (d, 2H), 4.35(m, 1H), 4.18-4.16 (t, 1H), 3.60-3.46 (m, 33H), 3.39-3.36 (t, 2H),2.50-2.23 (m, 2H), 2.23 (s, 3H), 2.23-1.93 (m, 2H), 1.48-1.23 (m, 6H),0.85-0.80 (m, 6H). LCMS: MH⁺1100, retention time 3.72 min.

4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190) (400 mg, 0.36 mmol) in DCM (10 mL) was added pyridine (0.14 mL,1.80 mmol), 4-nitrophenyl chloroformate (14) (145 mg, 0.72 mmol) at 0°C. The resultant reaction mixture was stirred at r.t for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 3% MeOH in DCM to get 0.34 g (75% yield) of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191) as a gum. LCMS: MH⁺1265, retention time1.33 min.

4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(192): To a stirred solution4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191) (281.73 mg, 0.22 mmol) in NMP (3.5 mL)was added TEA (0.07 Ml, 0.55 mmol), and(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) (100 mg, 0.22 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 8h. The progress of the reaction was monitored byLCMS. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with 10% methanol inchloroform (2×20 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. To the crudematerial diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography eluting with 4% MeOH in DCM to get 0.15 g (43% yield) of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(192) as a solid. LCMS: MH⁺1575, retention time 3.76 min.

4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(193): To a stirred solution of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(192) (150 mg, 0.09 mmol) in DCM 5 ml 1% TFA in DCM 2 ml was added at 0°C. and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain the desiredproducts (193) (32 mg, 25% yield) as a solid. ¹H NMR (400 MHz, DMSO-d₆):δ 9.98 (s, 1H), 8.06-8.11 (d, 2H), 7.88-7.90 (d, 1H), 7.56-7.59 (d, 2H),7.48-7.7.59 (d, 2H), 7.34 (s, 1H), 7.27-7.29 (d, 3H), 6.56 (s, 1H), 5.42(s, 2H), 5.29 (s, 2H), 4.92 (s, 2H), 4.20-4.22 (m, 4H), 3.47-3.601 (m,31H), 3.17-3.38 (m, 8H), 2.39-2.58 (m, 4H), 1.84-1.97 (m, 7H), 1.23-1.32(m, 7H), 0.81-0.89 (m, 9H), LCMS: MH⁺1319, retention time 1.53 min.

Example 32: Synthesis of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-35-(4-aminobutyl)-1-azido-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(200)

(S)-2-Amino-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(151): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)carbamate(150) (2 g, 2.74 mmol) in DMF (20 ml), 30% piperidine in DMF (4 ml) wasadded at r.t. The resultant reaction mixture was stirred at r.t for 2 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 80% EtOAc in hexane to get 1 g (72% yield)of(S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(151) as a pale brown gum. LCMS: MH⁺508, retention time 3.54 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(195): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-tryptophan (194) (301.59 mg, 0.70mmol), in DMF (10 mL) was added DIPEA (0.25 mL, 1.47 mmol), HATU (448.49mg, 1.18 mmol) and(S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(151) (300 mg, 0.59 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 0.3 g (55% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(195) as an off-white solid. LCMS: MH⁺916, retention time 2.40 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(196): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(195) (1 g, 1.09 mmol) in DCM (30 mL) was added pyridine (0.35 mL, 4.36mmol), 4-nitrophenyl chloroformate (105) (0.66 g, 3.27 mmol) at 0° C.The resultant reaction mixture was stirred at r.t for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by flashchromatography eluting with 1% MeOH in DCM to get 0.9 g (76% yield) of(9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(196) as a pale brown gum. LCMS: MH⁺1081, retention time 2.59 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(197): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(196) and (490.70 mg, 0.45 mmol) in DMSO (5 mL) was added TEA (0.10 Ml,0.75 mmol),(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) (170 mg, 0.37 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 8h. The progress of the reaction was monitored byLCMS. After completion of starting material, reaction mixture wasconcentrated under zen vac to get a crude residue. The crude waspurified by Combi-flash column chromatography eluting with 3% MeOH inDCM to get 0.42 g (79.8% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(197) as an off-white solid. LCMS: MH⁺1391, retention time 4.27 min.

4-((S)-2-((S)-2-Amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(198): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(197) (250 mg, 0.18 mmol) in DMF (5 ml) was treated with piperidine(0.05 ml, 0.53 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 2 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.20 mg (95% yield) of4-((S)-2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(198) crude as a sticky liquid and this material was used for the nextstep without purification.

4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(199): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (200mg, 0.17 mmol) in DMF (5 mL) was added DIPEA (0.07 mL, 0.42 mmol), HATU(130.06 mg, 0.34 mmol) and4-((S)-2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(198) (104.99 mg, 0.20 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 4% MeOH in DCM to get 0.13g (47% yield) of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(199) as a sticky solid. LCMS: MH⁺1662, retention time 3.77 min.

4-((32S,35S)-32-((1H-indol-3-yl)methyl)-35-(4-aminobutyl)-1-azido-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(200): To a stirred solution of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(11) (130 mg, 0.07 mmol) in DCM 5 ml 1% TFA in DCM 2 mL was added at 0°C. and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain the desiredproduct (200) (85 mg, 74.57% yield) as an off-white solid. ¹H NMR (400MHz, DMSO-d₆): (10.77 (s, 1H), 9.97 (s, 1H), 8.19-8.21 (d, 1H),8.07-8.09 (d, 1H), 8.00-8.02 (d, 1H), 7.56-7.60 (m, 4H), 7.48-7.51 (m,2H), 7.28-7.35 (m, 4H), 6.90-6.93 (m, 2H), 5.42 (s, 2H), 5.30 (s, 2H),4.96 (s, 2H), 4.41-4.58 (m, 1H), 4.40-4.57 (m, 1H), 4.23 (s, 2H),2.75-3.59 (m, 44H), 2.33-2.35 (d, 2H), 1.52-1.96 (m, 11H), 1.28-1.32 (t,6H), 0.85-0.89 (t, 3H), LCMS: MH⁺1406, retention time 2.06 min.

Example 33: Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(202)

4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201): To a stirred solution4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191) (311 mg, 0.25 mmol) in NMP (2.5 mL) wasadded TEA (0.09 mL, 0.62 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (131 mg, 0.25 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 8h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was quenched with water (15 mL) and extracted with 10% methanolin chloroform (2×20 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. To the crudematerial diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography eluting with 5% MeOH in DCM to get 0.300 g (78% yield) of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201) as a solid. LCMS: MH⁺1561, retention time 2.18 min.

4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(202): To a stirred solution of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201) (300 mg, 0.19 mmol) in DCM 5 ml 1% TFA in DCM was added at 0° C.and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain two peaksfor desired products (202) (70 mg) and the unwanted isomer (7 mg) as asolid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.96 (s, 1H), 8.12-8.10 (q, 2H),7.89-7.87 (d, 1H), 7.76-7.61 (d, 1H), 7.59-7.31 (m, 7H), 6.51 (s, 1H),5.44 (s, 2H), 5.29 (s, 3H), 5.09 (s, 2H), 4.37-4.20 (m, 1H), 4.18-4.16(t, 1H), 3.49-3.44 (m, 4H), 3.12-2.55 (m, 39H), 2.40-1.34 (m, 15H),0.89-0.82 (m, 9H), LCMS: MH⁺ 1305, retention time 5.33 and 5.47 min.

Example 34: Synthesis of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-35-(4-aminobutyl)-1-azido-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(206)

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] pyrano [3′,4′:6,7] indolizino [1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(203): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(196) (317.31 mg, 0.29 mmol) in DMSO (3 mL) was added TEA (0.06 mL, 0.48mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (16) (130 mg, 0.24 mmol) at 0° C.The resultant reaction mixture was stirred at r.t for 8h. The progressof the reaction was monitored by LCMS. After completion of startingmaterial, reaction mixture was concentrated under zen vac to get a cruderesidue. The crude was purified by Combi-flash column chromatographyeluting with 3% MeOH in DCM to get 0.28 g (83% yield) of(9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(203) as an off-white solid. LCMS: MH⁺1377, retention time 4.68 min.

4-((S)-2-((S)-2-Amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(204): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate(203) (280 mg, 0.20 mmol) in DMF (5 ml) was treated with piperidine(0.06 ml, 0.61 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 2 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.23 mg (99% yield) of4-((S)-2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(204) crude as a sticky liquid and this material was used for the nextstep without purification.

4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(205): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86)(53.13 mg, 0.10 mmol) in DMF (5 mL) was added DIPEA (0.03 mL, 0.21mmol), HATU (65.82 mg, 0.17 mmol) and4-((S)-2-((S)-2-amino-3-(1H-indol-3-yl)propanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(204) (100 mg, 0.08 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM to get0.0.08 g (56% yield) of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(205) as a sticky solid. LCMS: MH⁺ 1648, retention time 3.92 min.

4-((32S,35S)-32-((1H-indol-3-yl)methyl)-35-(4-aminobutyl)-1-azido-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(206): To a stirred solution of4-((32S,35S)-32-((1H-indol-3-yl)methyl)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(205) (80 mg, 0.04 mmol) in DCM 5 ml 1% TFA in DCM 2 mL was added at 0°C. and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain two peaksand Peak 1 is the desired product (206) (45 mg, 66% yield) and theunwanted isomer as Peak-2 (15 mg, 22% yield) as an off-white solid. ¹HNMR (400 MHz, DMSO-d₆): δ 10.77 (s, 1H), 10.00 (s, 1H), 9.96 (s, 1H),8.34 (s, 1H), 8.20-8.22 (d, 1H), 8.02-8.04 (d, 2H), 7.76-7.79 (d, 1H),7.56-7.63 (m, 3H), 7.28-7.38 (m, 4H), 7.13 (s, 1H), 7.00-7.04 (t, 1H),6.90-6.94 (s, 1H), 6.50 (s, 1H), 5.43 (s, 1H), 5.28 (s, 1H), 5.09 (s,1H), 4.39-4.57 (m, 2H), 4.01-4.03 (d, H), 3.31-3.59 (m, 38H), 2.94-2.95(m, 4H), 2.15-2.37 (m, 9H), 1.15-1.90 (m, 12H), 0.85-0.89 (t, 3H), LCMS:MH⁺1392, retention time 2.40 and 2.42 min.

Example 35: Synthesis of1-azido-N-((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(211)

(9H-Fluoren-9-yl)methyl((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(207): To a stirred solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid(76) (107.65 mg, 0.271 mmol) in DMF (5 mL) was added DIPEA (0.118 mL,0.677 mmol), HATU (171.671 mg, 0.451 mmol) and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (120 mg, 0.226 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was quenched with ice water. The precipitated solid wasfiltered off and dried under vacuum to get 0.150 g (81% yield) of(9H-fluoren-9-yl)methyl((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(207) as an off-white solid. This crude compound was used for the nextstep.

(R)-2-Amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(208): To a stirred solution of (9H-fluoren-9-yl)methyl((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(207) (150 mg, 184.20 mmol) in DMF (5 ml), 30% piperidine in DMF (1.5ml) was added at r.t. The resultant reaction mixture was stirred at r.tfor 1 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 100% EtOAc to get 0.100 g (91%yield) of(R)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(208) as a sticky liquid. This material was used for the next step.

(9H-Fluoren-9-yl)methyl((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(209): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (68.72 mg, 0.202mmol), in DMF (5 mL) was added DIPEA (0.074 mL, 0.422 mmol), HATU(128.32 mg, 0.337 mmol) and(R)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(208) (100 mg, 0.169 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 0.120 g (77% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(209) as a sticky liquid. LCMS: MH⁺914, retention time 3.14 min.

(R)-2-((S)-2-Amino-3-methylbutanamido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(210): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(209) (120 mg, 0.131 mmol) in DMF (5 mL), 30% piperidine in DMF (0.05ml) was added at r.t. The resultant reaction mixture was stirred at r.tfor 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 2.5% MeOH in DCM to get 0.110 g(98% yield) of(R)-2-((S)-2-amino-3-methylbutanamido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(210) as a sticky solid. LCMS: MH⁺692, retention time 2.44 min.

1-azido-N-((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(211): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86)(108.76 mg, 0.213 mmol) in DMF (5 mL) was added DIPEA (0.077 mL, 0.443mmol), HATU (134.73 mg, 0.354 mmol) and(R)-2-((S)-2-amino-3-methylbutanamido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(210) (105 mg, 0.177 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (10 mL) and extracted with EtOAc (2×15 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound wasinitially purified by flash chromatography eluting with 3% MeOH in DCMand then finally purified by RP-prep HPLC to obtain two peaks fordesired products (211) (Peak-1) (9 mg, 5% Yield) and an unwanted isomer(8 mg, 4% Yield) as an off-white sticky solid. ¹H NMR (400 MHz,DMSO-d₆): δ 8.37-8.39 (d, 1H), 8.01-8.03 (d, 1H), 7.78-7.84 (t, 2H),7.32 (s, 1H), 6.52 (s, 1H), 5.91 (s, 1H), 4.22-5.49 (m, 7H), 4.22 (s,1H), 4.08-4.11 (t, 1H), 3.32-3.59 (m, 39H), 2.92 (s, 2H), 1.33-2.32 (m,17H), 0.85-0.89 (t, 3H), 0.74-0.77 (t, 6H). LCMS: MH⁺ 1185, retentiontime 2.42 and 2.46 min.

Example 36: Synthesis ofN-((S)-1-(((S)-6-Amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(217)

(9H-Fluoren-9-yl)methyl((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)carbamate(212): To a stirred solution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (183.35 mg, 0.293 mmol) in DMF (5 mL) was added DIPEA (0.10 mL,0.611 mmol), HATU (185.97 mg, 0.489 mmol) and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (130 mg, 0.245 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was quenched with ice water. The precipitated solid wasfiltered off and dried under vacuum to get 0.200 g (78% yield) of(9H-fluoren-9-yl)methyl((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)carbamate(212) as an off-white solid. LCMS: MH⁺1042, retention time 2.67 min.

(S)-2-Amino-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(213): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)carbamate(212) (300 mg, 0.288 mmol) in DMF (5 ml), 30% piperidine in DMF (1.5 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for2 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 0.200 g (85% yield) of(S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(213) as a pale brown gum. LCMS: MH⁺820, retention time 3.88 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(214): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (152) (113.4 mg,0.293 mmol), in DMF (50 mL) was added DIPEA (0.10 mL, 0.61 mmol), HATU(185.48 mg, 0.488 mmol) and(S)-2-amino-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(213) (200 mg, 0.244 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 0.240 g (82% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(214) as an off-white solid. LCMS: MH⁺1189, retention time 4.84 min.

(S)-2-((S)-2-Amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(215): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(214) (200 mg, 0.168 mmol) in DMF (5 mL), 30% piperidine in DMF (0.05ml) was added at r.t. The resultant reaction mixture was stirred at r.tfor 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 2% MeOH in DCM to get 0.150 g (92%yield) of(S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(215) as a sticky solid. LCMS: MH⁺967, retention time 3.93 min

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(216): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86)(190.55 mg, 0.372 mmol) in DMF (5 mL) was added DIPEA (0.13 mL, 0.776mmol), HATU (236.05 mg, 0.621 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)hexanamide(215) (300 mg, 0.31 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (10 mL) and extracted with EtOAc (2×15 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to yield crude compound. The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM to get0.300 g (66% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(216) as a sticky solid. LCMS: MH⁺1460, retention time 3.86 min.

N-((S)-1-(((S)-6-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(217): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(216) (300 mg, 0.205 mmol) in DCM 5 ml 1% TFA in DCM (2 ML) and 1% TISin DCM (2 ML) were added at 0° C. and the resultant reaction mixture wasstirred at r.t for 1 h. The progress of the reaction was monitored byLCMS. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was triturated with diethyl ether and finaly purified byRP-prep-HPLC to obtain two peaks for desired product (217) (Peak-1) (23mg, 9% Yield) and an unwanted isomer Peak-2 was isolated. (17 mg, 7%Yield) as a brown sticky solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.51-8.53(d, 1H), 8.18-8.20 (d, 1H), 8.02-8.04 (d, 1H), 7.79-7.82 (d, 1H), 7.53(s, 2H), 7.32 (s, 1H), 7.19-7.22 (m, 5H), 6.53 (s, 1H), 5.12-5.50 (m,5H), 4.47 (s, 1H), 4.27 (s, 1H), 3.31-3.59 (m, 38H), 2.49-2.72 (m, 3H),1.33-2.30 (m, 18H), 0.85-0.88 (t, 3H). LCMS: MH⁺ 1204, retention time1.51 and 1.53 min.

Example 37: Synthesis of32-(((S)-1-(((S)-6-amino-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azapentatriacontan-35-oicacid (226)

(S)-2-((S)-2-Amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(218): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(153) (2 g, 2.28 mmol) in DMF (30 ml) was treated with piperidine (0.67ml, 6.84 mmol) and the reaction mixture stirred at 0° C. under nitrogenatmosphere for 1 h. The progress of the reaction was monitored by TLC.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound waspurified by flash chromatography eluting with 100% EtOAc to get 1.3 g(87% yield) of(S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(218) as a pale brown gum. LCMS: MH 655, retention time 1.87 min.

Allyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(220): To a stirred solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoicacid (219) (450.16 mg, 1.09 mmol) in DMF (20 mL) was added DIPEA (0.4mL, 2.29 mmol), HATU (696.75 mg, 1.83 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(218) (600 mg, 0.91 mmol) at r.t. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure and purify by combi-flash columnchromatography, to get 0.4 g (42% yield) of allyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(220) as a sticky solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.94 (s, 1H),8.13-8.8.15 (d, 1H), 7.93-7.95 (d, 1H), 7.87-7.89 (d, 2H), 7.68-7.71 (t,2H), 7.51-7.55 (t, 3H), 7.35-7.41 (m, 6H), 7.28-7.32 (t, 2H), 7.19-7.25(m, 9H), 7.11-7.15 (t, 4H), 7.02-7.04 (d, 2H), 5.81 (m, 1H), 5.24-5.29(d, 1H), 5.17-5.19 (d, 1H), 5.09-5.17 (t, 1H), 3.97-4.51 (m, 10H), 3.17(s, 3H), 3.00-3.01 (d, 1H), 2.21-2.39 (m, 8H), 1.17-1.98 (m, 9H).

Allyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(221): To a stirred solution of allyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(220) (500 mg, 0.47 mmol) in DCM (10 mL) was added pyridine (0.15 mL,1.91 mmol), 4-nitrophenyl chloroformate (25) (288.96 mg, 1.43 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 1.5% MeOH in DCM to get 0.4 g (69% yield) ofallyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(221) as a sticky liquid. LCMS: MH⁺1211, retention time 2.76 min.

Allyl4-amino-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(222): To a stirred solution of allyl4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(221) (410 mg, 0.33 mmol) in DMSO (5 mL) was added TEA (0.07 mL, 0.56mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (150 mg, 0.28 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 16h. The progress of thereaction was monitored by LCMS. After completion of starting material,reaction mixture was quenched with water (15 mL) and solid precipitatedout. It was filtered and the solid material was passed throughCombi-flash column chromatography eluting with 3% MeOH in DCM to get 0.3g (70% yield) of allyl4-amino-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(222) as an off-white solid. LCMS: MH⁺1285 retention time 2.22 min.

Allyl35-azido-4-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-6-oxo-9,12,15,18,21,24,27,30-octaoxa-5-azapentatriacontanoate(224): To a stirred solution of Azido-PEG9-Acid (86) (119.38 mg, 0.23mmol) in DMF (5 mL) was added DIPEA (0.08 mL, 0.48 mmol), HATU (147.89mg, 0.38 mmol) and allyl4-amino-5-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-5-oxopentanoate(223) (250 mg, 0.19 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×10 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM to get 0.34g (98% yield) of allyl35-azido-4-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-6-oxo-9,12,15,18,21,24,27,30-octaoxa-5-azapentatriacontanoate(224) as a sticky solid. LCMS: MH⁺1779, retention time 2.18 min.

1-azido-32-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azapentatriacontan-35-oicacid (225): To a stirred solution of allyl35-azido-4-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-6-oxo-9,12,15,18,21,24,27,30-octaoxa-5-azapentatriacontanoate(224) (200 mg, 0.11 mmol) in MTBE (6 mL) and water (3 ml) was addedmorpholine (0.49 mg, 0.006 mmol), Pd(P Ph₃)₄ (6.49 mg, 0.006 mmol) atr.t in presence of nitrogen atmosphere. The resultant reaction mixturewas stirred at r.t for 4 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 5% MeOH inDCM to get 0.16 g (82% yield) of1-azido-32-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azapentatriacontan-35-oicacid (225) as a sticky liquid. LCMS: MH⁺1739, retention time 1.61 min.

32-(((S)-1-(((S)-6-amino-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azapentatriacontan-35-oicacid (226): To a stirred solution of1-azido-32-(((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azapentatriacontan-35-oicacid (225) (150 mg, 0.08 mmol) in DCM 5 ml 1% TFA in DCM 2 ml was addedat 0° C. and the resultant reaction mixture was stirred at r.t for 1 h.The progress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain two peaks;the peak 1 was found to be desired product (226) (11 mg, 9% Yield) andan unwanted isomer was isolated as Peak 2 (9 mg, 7% yield) as anoff-white sticky solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.06 (s, 1H),8.44-8.40 (d, 2H), 8.20-822 (d, 1H), 8.04-8.09 (q, 2H), 7.75-7.78 (d,1H), 7.59-7.61 (d, 2H), 7.34-7.36 (d, 2H), 7.30 (s, 1H), 7.15-7.17 (m,5H), 6.51 (s, 1H), 5.44 (s, 2H), 5.27 (s, 2H), 5.07 (s, 2H), 4.27-4.52(m, 3H), 3.43-3.60 (m, 38H), 2.18-2.83 (m, 15H), 1.23-1.90 (m, 15H),0.85-0.89 (t, 3H), LCMS: MH⁺1482 retention time, 1.68 min.

Example 38: Synthesis of1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-1H-pyrrole-3-carboxamide(235)

(9H-fluoren-9-yl)methyl(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(77): (4-aminophenyl) methanol (75) (6 g, 4.87 mmol), Fmoc-Cit-OH (76)(23.24 g, 58.54 mmol), EEDQ (36.15 g, 146.34 mmol) were mixed in DCM-THF(1:1) (600 ml) and stirred at ambient temperature under nitrogen for 16h. The reaction mixture was reduced to dryness under vacuum and purifiedby column chromatography on a silica cartridge eluting with methanol/DCMgradient (5-10%). The solvent was evaporated under vacuum to obtain 11 g(45% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamatedesiredproduct (77) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.97 (s, 1H),7.90 (d, 2H), 7.73-7.76 (q, 2H), 7.65-7.67 (d, 1H), 7.54-7.56 (d, 2H),7.39-7.43 (t, 2H), 7.30-7.34 (m, 2H), 7.22-7.24 (d, 2H), 5.97-5.99 (t,1H), 5.41 (s, 1H), 5.07-5.10 (t, 1H), 4.42-4.43 (d, 2H), 4.14-4.13 (m,4H), 4.07-4.11 (q, 1H), 3.16-3.17 (d, 3H), 2.94-3.04 (m, 2H), 1.59-1.68(m, 2H), 1.38-1.47 (m, 2H). LCMS: MH⁺ 503, retention time 2.91 min.

(9H-fluoren-9-yl)methyl(S)-(1-((4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(227): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamatedesiredproduct (77) (2 g, 3.97 mmol) in Acetonitrile (20 ml) at 0° C. was addedNaI (1.78 g, 11.93 mmol) and TMSCl (1.52 mL, 11.93 mmol). The resultantreaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was extracted with EtOAc and water and collect theorganic layer dried over Na₂SO₄ and concentrated under vacuum. Then itwas purify under flash chromatography and dried under vacuum to get 2 g(82% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(227) as an off-white solid. LCMS: MH⁺613, retention time 3.44 min.

Methyl(S)-1-(4-(2-amino-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(229): To a stirred solution methyl 1H-pyrrole-3-carboxylate (228) (50mg, 0.4 mmol) in THF at 0° C. was added NaH (23.98 mg, 0.99 mmol) andfollowed by (9H-fluoren-9-yl)methyl(S)-(1-((4-(iodomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(227) (294 mg, 0.48 mmol). The resultant reaction mixture was stirred atr.t for 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was extracted withEtOAc and water and collect the organic layer dried over Na₂SO₄ andconcentrated under vacuum. Then it was purify under flash chromatographyand dried under vacuum to get 0.040 g (17% yield) of methyl(S)-1-(4-(2-amino-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(229) as a sticky brown solid. LCMS: MH⁺388, retention time 2.55 min.

Methyl1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(231): To a stirred solution of)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (26.27 mg, 0.077mmol), in DMF (5 mL) was added DIPEA (0.03 mL, 0.19 mmol), HATU (58.88mg, 0.15 mmol) and methyl(S)-1-(4-(2-amino-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(230) (30 mg, 0.07 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum at low temperature to get the crude compound.Then the crude compound was purified by flash chromatography to get0.030 g (55% yield) of methyl1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(231) as an off-white solid. LCMS: MH⁺709, retention time 3.24 min.

Methyl1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(232): To a stirred solution of (methyl1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(231) (600 mg, 0.98 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 10% MeOH in DCM to get 0.400 g (83% yield)of methyl1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(232) as a sticky solid and this material was used for the next step.

Methyl1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylate(233): To a stirred solution of Azido-PEG9-Acid (86) (504.65 mg, 0.98mmol), in DMF (5 mL) was added DIPEA (0.35 mL, 2.05 mmol), HATU (625.14mg, 1.64 mmol) and methyl1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1H-pyrrole-3-carboxylate(232) (400 mg, 0.82 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum at low temperature to get the crude compound.Then the crude compound was purified by flash chromatography to get0.500 g (62% yield) of methyl1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylate(233) as a brown liquid and this material was used for the next step.

1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylicacid (234): To a stirred solution ofmethyl1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylate(233) (0.450 g, 0.459 mmol) in Methanol:Water 10 ml (3:1), was addedLiOH·H₂O (0.193 g, 4.596 mmol) and stirred the reaction mixture atambient temperature for 72 h. After completion of starting material thereaction mixture was concentrated under vacuum and acidify by (1N) HCLto make the pH 7.0 and extracted by IPA in DCM for (5×10 ml) and solventwas evaporated to get the crude compound. The crude material waspurified by column chromatography on a silica cartridge eluting withmethanol/DCM gradient (10-15%). The solvent was evaporated under vacuumto obtain desired product 0.297 g (68% yield) of1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylicacid (234) as an off-white solid. LCMS: MH⁺966, retention time 2.41 min.

1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-1H-pyrrole-3-carboxamide(235): A solution of1-(4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl)-1H-pyrrole-3-carboxylicacid (234) (0.297 g, 0.308 mmol) and Exatecan mesylate (150 mg, 0.28mmol) in DMF (1 ml) was treated with HATU (213 mg, 0.561 mmol), andDIPEA (72 mg, 0.561 mmol) and stirred at ambient temperature undernitrogen atmosphere for 6 h. The reaction mixture was reduced to drynessunder vacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (5-10%). The solvent was evaporatedunder vacuum and the material was finally purified by RP prep-HPLC toobtain the desired compound as Peak-1 (235) as an off-white solid (50mg, 13% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.98-9.86 (s, 2H), 8.4-8.1(m, 4H), 7.8 (d, 1H), 7.7 (d, 1H), 7.6 (d, 1H), 7.2 (s, 1H), 7.1 (d,2H), 6.8-6.4 (s, 3H), 5.93-5.69 (m, 2H), 5.39 (d, 2H), 5.21 (d, 2H),5.07 (s, 2H), 3.47-3.22 (m, 23H), 2.96 (m, 4H), 2.6-2.1 (m, 7H),1.89 (m,3H), 1.85-1.14 (m, 6H), 0.808 (m, 6H).LCMS: MH⁺1384, retention time 5.87min.

Example 39: Synthesis of4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(241)

(9H-fluoren-9-yl)methyl(S)-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(237): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)glycine (236) (100 mg, 0.336 mmol),in DMF (5 mL) was added DIPEA (0.14 mL, 0.841 mmol), HATU (255 mg, 0.673mmol) and(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) (226 mg, 0.505 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under reduced pressure at low temperature. The crude compoundwas purified by flash chromatography eluting with 5% MeOH in DCM to get170 mg (79% yield) of (9H-fluoren-9-yl)methyl(S)-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(237) as an off-white solid. LCMS: MH⁺ 729, retention time 3.02 min.

(S)-2-amino-N-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)acetamide(238): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(237) (170 mg, 0.233 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 20% MeOH in DCM to get 110 mg (93% yield) of(S)-2-amino-N-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)acetamide(238) as a white solid. LCMS: MH⁺507, retention time 2.43 min

4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(239): To a stirred solution of(S)-2-amino-N-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)acetamide(238) (110 mg, 0.237 mmol) in DMSO (2 ml), TEA (0.14 mL, 0.949 mmol),and then (9H-fluoren-9-yl)methyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((((Z)-penta-1,3-dien-2-yl)oxy)carbonyl)oxy)methyl)phenyl)amino)-5-ureidopentan-2-yl)amino)butan-2-yl)carbamate(81) (544 mg, 0.711 mmol) was added at r.t. The resultant reactionmixture was stirred at r.t for 1h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was concentrated under Gen-Vac to get crude compound. The crudecompound was purified by flash chromatography eluting with 10% MeOH inDCM to get 200 mg (72% yield) of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(239) as an off-white solid. LCMS: MH⁺1134, retention time 4.08 min.

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(240): To a stirred solution of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(239) (200 mg, 0.176 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at r.t. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 20% MeOH in DCM to get 100 mg (93% yield) of4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(240) as a white solid. LCMS: MH⁺912, retention time 2.57 min

4-((32S,35S)-1-azido-32-isopropyl-30,33-dioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(241): To a stirred solution of Azido-PEG9-Acid (86) (40 mg, 0.078mmol), in DMF (5 mL) was added DIPEA (0.14 mL, 0.196 mmol), HATU (255mg, 0.157 mmol) and4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(2-((3-(((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)amino)-2-oxoethyl)carbamate(240) (106 mg, 0.117 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 4 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasreduced to dryness under vacuum and purified by column chromatography ona silica cartridge eluting with methanol/DCM gradient (1-2%). Thesolvent was evaporated under vacuum and the material was finallypurified by RP prep-HPLC to obtain desired product (241) (8 mg, 7%) as asticky solid. LCMS: MH⁺1405, retention time 2.38 min. ¹H NMR (400 MHz,DMSO-d₆): δ 9.96 (s, 1H), 8.35 (d, 2H), 8.01 (t, 2H), 7.71-7.67 (d, 1H),7.59-7.31 (m, 7H), 6.48 (s, 1H), 5.95 (s, 1H), 5.87 (s, 2H) 5.42-5.31(6, 3H), 4.93 (s, 2H), 4.60 (t, 1H), 4.34 (m, 1H), 4.22 (s, 2H), 3.86(d, 2H), 3.62 (d, 2H), 3.69-3.01 (m, 36H), 2.66 (m, 1H), 2.35 (t, 2H),1.98 (m, 2H), 1.87 (m, 4H), 1.68-1.53 (t, 4H) 1.18 (t, 3H) 1.31 (t, 3H),0.81 (d, 6H) LCMS: MH⁺1405, retention time 2.38 min

Example 40: Synthesis of4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9s)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(251) (TFA salt)

4-((tert-butyldimethylsilyl)oxy)aniline (243): To a stirred solution of4-aminophenol (242) (4.0 g, 36.65 mmol), in DCM (40 mL) was addedimidazole (3.74 g, 54.98 mmol) followed by TBDMS-C₁ (6.08 g, 40.32 mmol)under nitrogen atmosphere at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture was cooledto 0° C., quenched with ice cold water (80 mL), and extracted with DCM(2×100 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 10% EtOAc in pet ether to get 4.0 g (49% yield) of4-((tert-butyldimethylsilyl) oxy) aniline (242) as a pale brown semisolid. LCMS: m/z 224.18 [(M+H)⁺]; R_(t): 1.47 min; 99.88% purity.

(9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(244): To a stirred solution of 4-((tert-butyldimethylsilyl)oxy)aniline(243) (2.0 g, 8.95 mmol) in DMF was added(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid(76) (3.56 g, 8.95 mmol), DIPEA (4.70 mL, 26.85 mmol) and HATU (5.10 g,13.43 mmol) at r.t under nitrogen atmosphere The reaction mixture wasstirred at r.t for 4h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried to afford crude compound. The crude compound was triturated withdiethyl ether (30 mL) to get 3.0 g (56% yield) of(9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(244) as an off-white solid. LCMS: m/z 603.41 [(M+H)⁺]; R_(t): 2.23 min;82.21% purity.

(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(245): To a stirred solutionof(9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(244) (3.0 g, 4.98 mmol) in DMF (10 mL) was added 30% piperidine in DMF(1.0 mL) under nitrogen atmosphere at r.t. The resultant reactionmixture was stirred at r.t for 2h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was concentrated under reduced pressure to get crude compound.The crude compound was purified by column chromatography using silicagel (100-200 mesh) eluting with 20% MeOH in DCM to get 1.8 g (95% yield)of(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(245) as an off-white solid. LCMS: m/z 381.53 [(M+H)⁺]; R_(t): 1.58 min;85.59% purity.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(246): To a stirred solution of(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(245) (2.0 g, 5.26 mmol) in DMF (20 mL) was added EDC.HCl (2.02 g, 10.52mmol), HOAt (1.07 g, 7.89 mmol) and(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (1.78 g, 5.26 mmol)at 0° C. The resultant reaction mixture was stirred at r.t for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quench with ice water. Theprecipitated solid was filtered off and dried under vacuum to get crudecompound. The crude compound was triturated with diethyl ether (30 mL)to get 1.5 g (41% yield) of(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(246) as an Off-white solid. LCMS: m/z 702.65 [(M+H)⁺]; R_(t): 2.49 min;78.61% purity.

(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(247): To a stirred solution of(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(246) (2.0 g, 2.85 mmol) in DMF (20 mL) was added 30% piperidine in DMF(2.0 mL) under nitrogen atm at r.t. The resultant reaction mixture wasstirred at r.t for 2h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 20% MeOH in DCM to get 800 mg (crude) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(247) as an off-white solid. LCMS: m z 480.40 [(M+H)⁺]; R_(t): 1.67 min;31.52% purity.

1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(248): To a stirred solution of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-ureidopentanamide(247) (700 mg, 1.46 mmol) in DMF (10 mL) was added DIPEA (0.76 mL, 4.38mmol), PyBOP (1.14 g, 2.19 mmol) and1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) (553 mg, 1.46mmol) at 0° C. to rt. The resultant reaction mixture was stirred at r.tfor 3 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quench with icewater and extracted with EtOAc (2*50 mL) to get crude compound. Thecrude compound was triturated with diethyl ether (30 mL) to get 500 mg(41% yield) of1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(248) as an off-white solid. LCMS: m/z 841.66 [(M+H)⁺]; R_(t): 2.13 min;44.34% purity.

1-azido-N-((S)-1-(((S)-1-((4-hydroxyphenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(249): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(248) (300 mg, 0.36 mmol) in methanol (10 mL) was added NH₄F (133 mg,3.6 mmol) under nitrogen atm at r.t. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure, quenched with ice water andextracted with 10% methanol in DCM (2×100 mL) to afford crude compound.The crude compound was triturated with diethyl ether, filtered and driedunder vacuum to furnish 200 mg (77% yield) of1-azido-N-((S)-1-(((S)-1-((4-hydroxyphenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(249) as an off-white solid. LCMS: m z 727.56 [(M+H)⁺]; R_(t): 1.55 min;95.26% purity.

4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl) carbonate (250): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-hydroxyphenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(249) (200 mg, 0.28 mmol) in DCM (5 mL) was added triethylamine (0.12mL, 1.64 mmol), 4-nitrophenyl carbonochloridate (25) (132 mg, 0.65 mmol)and catalytic amount of DMAP at 0° C. The resultant reaction mixture wasstirred at r.t for 4 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (230-400mesh) eluting with 3% MeOH in DCM to get 0.150 g (61% yield) of4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl) carbonate (250) as a pale brown solid. LCMS: m/z 892.62[(M+H)⁺]; R_(t): 1.83 min; 92.32% purity.

4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(251) TFA salt: To a stirred solution of4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl)carbonate (250) (100 mg, 0.11 mmol) in DMF (5 mL) was added pyridine (26mg 0.33 mmol), HOBt (30 mg, 0.22 mmol) and(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (48 mg, 0.11 mmol) at 0° C. to rt. The resultantreaction mixture was stirred at r.t for 16 h. The progress of thereaction was monitored by TLC. After completion of starting material,after completion of starting material, reaction mixture was quenchedwith ice water and extracted with 10% MeOH/DCM (2×30 mL) to afford crudecompound. The crude compound was purified by RP-Prep HPLC and thepurified fractions were lyophilized to furnish 12.4 mg (9% yield)4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(251) (TFA salt) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ10.02 (s, 1H), 8.54 (d, J=8.9 Hz, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.84 (q,J=12.6 Hz, 2H), 7.64 (d, J=8.8 Hz, 2H), 7.33 (s, 1H), 7.22 (d, J=8.8 Hz,2H), 6.52 (br. s, 1H), 5.98 (s, 1H), 5.45-5.30 (m, 7H), 4.39 (d, J=5.9Hz, 1H), 4.24 (t, J=7.6 Hz, 1H), 3.60-3.42 (m, 24H), 3.18 (d, J=6.8 Hz,4H), 3.0-2.96 (m, 2H), 2.40-2.33 (m, 7H), 1.99-1.85 (m, 3H), 1.65 (t,J=23.0 Hz, 2H), 1.41 (t, J=7.6 Hz, 2H), 0.90-.083 (m, 9H). LC-MS (method25): m/z 1188.83 [(M+H)⁺]; R_(t): 1.80 min; 97.80% purity, HP-LC (method25): R_(t): 3.89 min; 98.18% purity.

Example 41: Synthesis of4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(260) (TFA salt)

(9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(252): To a stirred solution of 4-((tert-butyldimethylsilyl)oxy)aniline(243) (1.2 g, 5.37 mmol) in DMF (30 mL) was addedN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (3.36 g, 5.37 mmol), DIPEA (2.8 mL, 16.11 mmol) and PyBOP (4.2 g,8.06 mmol) at rt under nitrogen atmosphere The resultant reactionmixture was stirred at r.t for 3h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was quenched with ice water to get precipitated, filtered thesolid and dried to get crude compound. The crude compound was trituratedwith n-pentane (30 mL) to get 3.3 g (74% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(252) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.9 (s, 1H),7.89-7.86 (m, 2H), 7.71 (t, J=6.8 Hz, 2H), 7.59 (d, J=8.0 Hz, 1H), 7.46(d, J=8.8 Hz, 2H), 7.37 (d, J=8.0 Hz, 5H), 7.29-7.22 (m, 7H), 7.13 (t,J=7.2 Hz, 2H), 7.04 (d, J=8.0 Hz, 2H), 6.78 (d, J=8.8 Hz, 1H), 4.29-4.06(m, 2H), 3.03-2.98 (m, 4H), 2.88 (s, 1H), 2.73 (s, 1H), 2.49 (t, J=1.6Hz, 1H), 2.22 (s, 1H), 1.95 (d, J=6.8 Hz, 2H), 1.74-1.71 (m, 2H),1.50-1.46 (m, 5H), 0.93 (s, 9H), 0.15 (s, 6H).

(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(253): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(252) (3.3 g, 3.98 mmol) in DMF (33 mL) was added 30% piperidine in DMF(3.3 mL) under nitrogen atm at rt. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 55% EtOAc in pet ether to get 2.4 g (99% yield) of(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(253) as an pale brown solid. LCMS: m/z 608.50 [(M+H)⁺]; R_(t): 2.18min; 81.13% purity.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(254): To a stirred solution of(S)-2-amino-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(253) (2.4 g, 3.95 mmol) in DMF (24 mL) was added DIPEA (2.1 mL, 11.85mmol), HATU (2.25 g, 5.93 mmol) and(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (1.34 g, 3.95 mmol)at 0° C. The resultant reaction mixture was stirred at r.t for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with ice water. Theprecipitated solid was filtered off, dried to get crude compound. Thecrude compound was triturated with diethyl ether (30 mL) to afford 2.5 g(68% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(254) as an off-white solid. LCMS: m/z 705.76 [(M−H-Fmoc)-]; R_(t): 2.97min; 87.14% purity.

(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(255): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(254) (2.5 g, 2.69 mmol) in DMF (25 mL) was added 30% piperidine in DMF(2.5 mL) under nitrogen atm at rt. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 80% EtOAc in pet ether to get 1.5 g (79% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(255) as a pale brown semi solid. LCMS: m z 707.56 [(M+H)⁺]; R_(t): 2.0min; 91.46% purity.

1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(256): To a stirred solution of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(255) (559 mg, 0.79 mmol) in DMF (5 mL) was added DIPEA (0.4 mL, 2.37mmol), PyBOP (617 mg, 1.19 mmol) and1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) (300 mg, 0.79mmol) at 0° C. to rt. The resultant reaction mixture was stirred at r.tfor 3 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quenched with icewater and extracted with EtOAc (2×30 mL). Combined organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford the crude compound. The crude compound was trituratedwith diethyl ether (30 mL) to get 300 mg (35% yield) of1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(256) as an off-white solid. LCMS: m/z 1069.13 [(M+H)⁺]; R_(t): 3.02min; 93.69% purity.

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-hydroxyphenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(257): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-((tert-butyldimethylsilyl)oxy)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(256) (500 mg, 0.47 mmol) in methanol (10 mL) was added NH₄F (174 mg,4.7 mmol) under nitrogen atm at rt. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure and quenched with ice water,extracted with 10% methanol in DCM (2×100 mL). Combined organic layerwas dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford the crude compound. The crude compound was trituratedwith diethyl ether (30 mL) to get 300 mg (67% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-hydroxyphenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(257) as an off-white solid. LCMS: m/z 954.97 [(M+H)⁺]; R_(t): 1.84 min;90.94% purity.

4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl) carbonate (258): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-hydroxyphenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(257) (120 mg, 0.13 mmol) in DCM (5 mL) was added triethyl amine (0.05mL, 0.39 mmol), 4-nitrophenyl carbonochloridate (16) (52 mg, 0.26 mmol)and catalytic amount of DMAP (5 mg) at 0° C. The resultant reactionmixture was stirred at r.t for 4 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was concentrated under reduced pressure to get 90 mg (crude) of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl) carbonate (258) as a pale brown solid. The crudecompound was used in the next step without any further purification.LCMS: m z 1120.14 [(M+H)⁺]; R_(t): 2.23 min; 40.91% purity.

4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(259): To a stirred solution of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl(4-nitrophenyl) carbonate (258) (100 mg, 0.09 mmol) in DMF (2 mL) wasadded pyridine (26 mg 0.33 mmol), HOBt (18 mg, 0.14 mmol), and(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(16) (39 mg, 0.09 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water and extracted with 10% MeOH/DCM (2×30 mL).Combined organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford 90 mg (crude) of3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(259) as an off-white solid. The crude compound was used in the nextstep without any further purification. LC-MS: m z 1416.42 [(M+H)⁺];R_(t): 2.23 min; 79.07% purity.

4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(260) (TFA salt): To a stirred solution of3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(259) (90 mg, 0.063 mmol) in DCM (4 mL) was added 1% TFA in DCM at 0° C.The resultant reaction mixture was stirred at r.t for 1 h. The progressof the reaction was monitored by LCMS. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by RP-prep HPLC tofurnish 11 mg (15% yield) of4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(260) (TFA salt) as an off-white solid. ¹H NMR (400 MHz, DMSO): δ 10.03(s, 1H), 8.55 (d, J=8.7 Hz, 1H), 8.15 (d, J=7.7 Hz, 1H), 7.91 (d, J=8.3Hz, 1H), 7.81 (d, J=10.9 Hz, 1H), 7.65-7.61 (m, 4H), 7.34 (s, 1H), 7.24(d, J=8.9 Hz, 2H), 6.53 (s, 1H), 5.42-5.30 (m, 5H), 4.38 (d, J=6.0 Hz,1H), 4.21 (t, J=7.6 Hz, 1H), 3.60-3.48 (m, 24H), 3.38 (t, J=5.0 Hz, 2H),3.17 (d, J=4.8 Hz, 2H), 2.78 (s, 2H), 2.37-2.32 (m, 6H), 2.20 (d, J=8.8Hz, 1H), 1.98-1.86 (m, 3H), 1.69 (m, 2H), 1.54 (q, J=7.5 Hz, 2H), 1.37(d, J=10.8 Hz, 2H), 0.90-0.83 (m, 9H). LC-MS (method 30): m/z 1159.45[(M+H)⁺]; R_(t): 2.12 min; 94.33% purity, HP-LC (method 30): R_(t): 3.66min; 93.22% purity.

Example 42:4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(269) (TFA salt)

Methyl(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(262): To a stirred solution ofN²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(diphenyl(p-tolyl)methyl)-L-lysine(149) (2.0 g, 3.20 mmol) in DMF (20 mL) was added methyl 4-aminobenzoate(261) (484 mg, 3.20 mmol), HATU (2.43 g, 6.40 mmol) and DIPEA (1.68 mL,9.60 mmol) at rt. The resultant reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was quenched with ice water (50mL) and the precipitated solid was filtered off, dried under vacuum toget crude compound. The crude compound was purified by columnchromatography using silica gel (100-200 mesh) eluting with 20-25% EtOAcin Pet ether to get 700 mg (29% Yield) of methyl(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(262) as an off-white solid. LC-MS: m z 758.70 [(M+H)⁺]; R_(t): 2.96min; 71.01% purity.

Methyl(S)-4-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(263): To a stirred solution of methyl(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(262) (700 mg, 0.92 mmol) in DMF (5 mL) was added piperidine (0.7 mL)under nitrogen atm at rt. The resultant reaction mixture was stirred atr.t for 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby column chromatography using silica gel (100-200 mesh) eluting with2-3% MeOH in DCM to get 490 mg (99% Yield) of methyl(S)-4-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(263) as a pale yellow solid. LC-MS: m z 536.66 [(M+H)⁺]; R_(t): 2.61min; 69.45% purity.

Methyl4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(264): To a stirred solution of methyl(S)-4-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(264) (480 mg, 0.90 mmol) in DMF (5 mL) was added(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (305 mg, 0.90 mmol),HATU (513 mg, 1.35 mmol) and DIPEA (0.47 mL, 2.70 mmol) at rt. Theresultant reaction mixture was stirred at r.t for 2 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with ice water (20 mL) and theprecipitated solid was filtered off, dried under vacuum to get crudecompound. The crude compound was triturated with diethyl ether (20 mL)and filtered off, dried under vacuum to get 700 mg (91% yield) of methyl4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(264) as a pale yellow solid. LC-MS: m z 857.79 [(M+H)⁺]; R_(t): 2.93min; 49.65% purity.

Methyl4-((S)-2-((S)-2-amino-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate (265): To a stirred solution of methyl4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(264) (700 mg, 0.82 mmol) in DMF (5 mL) was added piperidine (0.70 mL)under nitrogen atm at rt. The resultant reaction mixture was stirred atr.t for 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby column chromatography using silica gel (100-200 mesh) eluting with2-3% MeOH in DCM to get 350 mg (67% yield) of methyl4-((S)-2-((S)-2-amino-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(265) as a yellow gum. LC-MS: m z 633.79 [(M−H)-]; R_(t): 2.64 min;76.44% purity.

Methyl4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoate(266): To a stirred solution of methyl4-((S)-2-((S)-2-amino-3-methylbutanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzoate(265) (200 mg, 0.32 mmol) in DMF (3 mL) was added1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (119) (121 mg, 0.32mmol), PyBOP (250 mg, 0.48 mmol) and DIPEA (0.17 mL, 0.96 mmol) at 0° C.The resultant reaction mixture was stirred at r.t for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with ice water (30 mL) and theprecipitated solid was filtered off, dried under vacuum to get crudecompound. The crude compound was triturated with diethyl ether (20 mL)and filtered off, dried under vacuum to get 300 mg (96% yield) of methyl4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoate(266) as a pale yellow solid. LC-MS: m z 995.18 [(M−H)-]; R_(t): 2.65min; 75.58% purity.

4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoicacid (267): To a stirred solution of methyl4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoate(266) (250 mg, 0.25 mmol) in THF (3 mL) was added 0.5N NaOH (2.5 mL) at0° C. The resultant reaction mixture was stirred at r.t for 48 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure and water (20 mL) was added followed by acidification withsaturated citric acid solution and the precipitated solid was filteredoff, dried under vacuum to get crude compound. The crude compound wastriturated with diethyl ether (20 mL) and filtered off, dried undervacuum to get 150 mg (610% yield) of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoicacid (267) as a pale yellow solid. LC-MS: m z 981.16 [(M−H)⁻]; R_(t):2.00 min; 62.86% purity.

4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(268): To a stirred solution of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzoicacid (267) (140 mg, 0.14 mmol) in DMF (4 mL) was added(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (74 mg, 0.14 mmol), HATU (80 mg, 0.21 mmol) andDIPEA (0.07 mL 0.42 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water (10 mL) and the precipitated solid was filteredoff, dried under vacuum to get crude compound. The crude compound wastriturated with diethyl ether (20 mL) and filtered off, dried undervacuum to get 120 mg (60% yield) of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(268) as a pale brown solid. LC-MS: m z 1399.94 [(M+H)⁺]; R_(t): 2.16min; 76.27% purity.

4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(269) (TFA salt): To a stirred solution of4-((23S,26S)-1-azido-26-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(268) (110 mg, 0.08 mmol) in DCM (5 mL) was added 1% TFA in DCM (11 mL)at 0° C. The resultant reaction mixture was stirred at r.t for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified byRP-prep HPLC to afford 15.5 mg (17% yield) of4-((23S,26S)-26-(4-aminobutyl)-1-azido-23-isopropyl-21,24-dioxo-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)benzamide(269) (TFA salt) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ10.19 (s, 1H), 8.91 (d, J=8.2 Hz, 1H), 8.16 (d, J=7.6 Hz, 1H), 7.92-7.90(m, 3H), 7.82 (d, J=10.8 Hz, 1H), 7.69-7.62 (m, 5H), 7.31 (s, 1H), 6.52(s, 1H), 5.81-5.72 (m, 1H), 5.37 (s, 2H), 5.16 (q, J=19.9 Hz, 2H),4.39-4.32 (m, 1H), 4.19 (t, J=7.4 Hz, 1H), 3.60-3.38 (m, 27H), 2.78-2.72(m, 2H), 2.42-2.26 (m, 7H), 1.96-1.52 (m, 7H), 1.41-1.31 (m, 2H),0.87-0.82 (m, 9H). LC-MS (method 31): m z 1143.87 [(M+H)⁺]; R_(t): 1.68min; 97.58% purity, HP-LC (method-31): R_(t): 3.53 min; 97.23% purity.

Example 43: Synthesis of4-((32S,35S)-35-(4-Aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(280)

4-(((Tert-butyldiphenylsilyl)oxy)methyl)-2-methylaniline (271): To astirred solution of (4-amino-3-methylphenyl)methanol (270) (1.5 g,10.935 mmol) in DMF (15 mL) was added imidazole (1.489 g, 21.869 mmol)followed by tert-butyl(chloro)diphenylsilane (3.607 g, 13.121 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with water (15 mL) andextracted with EtOAc (2×100 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to getcrude compound. The crude compound was purified by flash chromatographyeluting with 15% EtOAc in hexane to get 2 g (48% yield) of4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylaniline (271) as a palebrown gum. LCMS: MH⁺ 376, retention time 4.54 min.

(9H-Fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(272): To a stirred solution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (1.164 g, 1.864 mmol) in DMF (10 mL) was added DIPEA (0.96 mL,5.592 mmol), HATU (1.41 g, 3.728 mmol) and4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylaniline (271) (700 mg,1.864 mmol) at 0° C. The resultant reaction mixture was stirred at r.tfor 16 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quenched with icewater. The precipitated solid was filtered off and dried under vacuum toget 1.2 g (65% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(272) as an off-white solid. LCMS: MH⁺982, retention time 3.58 min.

(S)-2-Amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(273): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(272) (1.5 g, 1.529 mmol) in DMF (15 ml), 30% piperidine in DMF (1 ml)was added at rt. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 0.800 g (68% yield) of(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(273) as a pale brown gum. LCMS: MH⁺760, retention time 5.07 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(274): To a stirred solution of(((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (152) (1.27 g,3.292 mmol), in DMF (20 mL) was added DIPEA (1.70 mL, 9.875 mmol), HATU(2.50 g, 6.583 mmol) and(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(273) (2.5 g, 3.292 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 3.1 g (83% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(274) as an off-white solid. LCMS: MH⁺1129, retention time 5.04 min.

(S)-2-((S)-2-Amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(275): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(274) (3.1 g, 2.747 mmol) in DMF (10 mL), 30% piperidine in DMF (0.39ml) was added at rt. The resultant reaction mixture was stirred at r.tfor 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 100% EtOAc to get 2 g (80% yield)of(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(275) as an off-white solid. LCMS: MH⁺907, retention time 3.80 min.

1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(276): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86)(412.19 mg, 0.806 mmol) in DMF (10 mL) was added DIPEA (0.41 mL, 2.417mmol), HATU (459.55 mg, 1.209 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(275) (730 mg, 0.806 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 3.5% MeOH in DCM to get0.370 g (33% yield) of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(276) as a pale brown gum. LCMS: MH⁺1400, retention time 3.49 min.

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methylphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(277): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methylphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(276) (730 mg, 0.522 mmol) in methanol (10 mL) was added NH₄F (193.26mg, 5.218 mmol) at rt. The resultant reaction mixture was stirred at r.tfor 6 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get a crude residue. The residue obtained wasdiluted with water (15 mL) and extracted with EtOAc (2×20 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound waspurified by flash chromatography eluting with 5% MeOH in DCM to get0.370 g (61% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methylphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(277). LCMS: MH⁺1162, retention time 3.85 min.

4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl(4-nitrophenyl) carbonate (278): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methylphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(279) (350 mg, 0.301 mmol) in DCM (10 mL) was added pyridine (0.12 mL,1.507 mmol), 4-nitrophenyl chloroformate (25) (182.28 mg, 0.904 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 4% MeOH in DCM to get 0.220 g (55% yield) of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl(4-nitrophenyl) carbonate (278) as a pale brown gum. LCMS: MH⁺1327,retention time 2.24 min.

4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(279): To a stirred solution of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl(4-nitrophenyl) carbonate (278) (200 mg, 0.151 mmol) in NMP (3 mL) wasadded TEA (0.05 mL, 0.377 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (65.60 mg, 0.151 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 4h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was quenched with water (15 mL) and extracted with 10% methanolin chloroform (2×20 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. To the crudematerial diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography eluting with 6.5% MeOH in DCM to get 0.140 g (57% yield)of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(279) as an off-white solid. LCMS: MH⁺1624, retention time 2.11 min.

4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methylbenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(280): To a stirred solution of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(279) (140 mg, 0.086 mmol) in DCM 5 ml 1% TFA in DCM was added at 0° C.and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain the desiredproduct (280) (26 mg, 22% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 9.31 (s, 1H), 8.26-8.28 (q, 1H), 8.01-8.10 (q, 2H),7.71-7.79 (d, 1H), 7.61 (s, 3H), 7.37-7.39 (d, 1H), 7.31 (s, 1H),7.15-7.26 (m, 6H), 6.51 (s, 1H), 5.42 (s, 2H), 5.08 (s, 3H), 4.56 (s,2H), 4.56 (s, 1H), 4.44-4.56 (d, 1H), 3.01-3.60 (m, 36H), 2.78-2.80 (m,3H), 2.17-2.49 (m, 9H), 1.23-1.88 (m, 10H), 0.85-0.88 (t, 3H). LCMS: MH⁺1367, retention time 1.96 and 1.98 min.

Example 44:(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(293) (TFA salt)

1-(4-nitrophenyl)-1H-imidazole-2-carbaldehyde (282): To a stirredsolution of 1-fluoro-4-nitrobenzene (281) (5.0 g, 35.44 mmol) and1H-imidazole-2-carbaldehyde, (3.41 g, 35.44 mmol) in dry DMF (50 mL) wasadded K₂CO₃ (6.37 g, 46.07 mmol) at rt under nitrogen atmosphere Theresultant reaction mixture was heated to 80° C. for 6 h. The progress ofthe reaction was monitored by TLC. After completion of reaction, thereaction mixture was allowed to cool to room temperature and water (250mL) was added with vigorous stirring. The resultant precipitate wasfiltered, washed with water (3×70 mL) and dried under vacuum to afford6.0 g (78% yield) of 1-(4-nitrophenyl)-1H-imidazole-2-carbaldehyde (282)as pale brown solid. LC-MS: m z 218.18 [(M+H)⁺]; R_(t): 1.49 min; 96.10%purity.

(1-(4-nitrophenyl)-1H-imidazol-2-yl)methanol (283): To a stirredsolution of 1-(4-nitrophenyl)-1H-imidazole-2-carbaldehyde (282) (6.0 g,27.63 mmol) in MeOH (120 mL) was added NaBH₄ (575 mg, 15.19 mmol)portion wise at 0° C. The resultant reaction mixture was stirred at 0°C. for 1 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was quenched withice water (50 mL). The mixture was concentrated to approximately 20% ofthe original volume and additional water (150 mL) was added. Theprecipitated solid was filtered, washed with water dried under vacuum toafford 5.6 g (92% Yield) of (1-(4-nitrophenyl)-1H-imidazol-2-yl)methanol(283) as a grey solid. LC-MS: m z 219.99 [(M+H)⁺]; R_(t): 0.75 min;96.81% purity.

2-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4-nitrophenyl)-1H-imidazole(284): To a stirred solution of(1-(4-nitrophenyl)-1H-imidazol-2-yl)methanol (283) (5.6 g, 25.55 mmol)in DCM (60 mL) was added imidazole (3.48 g, 51.09 mmol) and cooled to 0°C. TBDMS-C₁ (5.04 g, 33.21 mmol) was added portion wise to the reactionmixture, stirred at room temperature for 8 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was diluted with DCM, washed with water. The organiclayer was dried over anhydrous Na₂SO₄, concentrated under reducedpressure to get crude compound. The crude compound was purified by usingcolumn chromatography on silica gel 100-200 mesh eluting with 15-20%ethyl acetate in pet ether to get 6.5 g (76% yield) of2-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4-nitrophenyl)-1H-imidazole(284) as pale yellow solid. LC-MS: m z 334.23 [(M+H)⁺]; R_(t): 1.95 min;99.38% purity.

4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)aniline(285): To a stirred solution of2-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4-nitrophenyl)-1H-imidazole(284) (3.5 g, 10.50 mmol) in MeOH (35 mL) was added 10% Pd/C (350 mg).The resultant reaction mixture was stirred at room temperature under H₂balloon pressure for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasfiltered through celite bed, washed with methanol, dried under vacuum to3.0 g (94% yield) of4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)aniline(285) as colorless liquid. LC-MS: m z 304.30 [(M+H)⁺]; R_(t): 1.53 min;99.34% purity.

(9H-fluoren-9-yl)methyl(S)-(1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(286): To a stirred solution of4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)aniline(285) (1.0 g, 3.29 mmol) and(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid(76) (1.31 g, 3.29 mmol) in DMF (10 mL) was added EEDQ (1.22 g, 4.94mmol)) at 0° C. The resultant reaction mixture was stirred at roomtemperature for 16 h. The progress of the reaction was monitored by TLC.After completion of starting material, the reaction mixture was quenchedwith ice water (50 mL). The precipitate was gummy in nature. Water wasdecanted off and the gummy compound was washed with water and diethylether to afford 1.0 g (crude) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(286) as a pale yellow gum. LC-MS: m z 683.40 [(M+H)⁺]; R_(t): 1.92 min;35.15% purity.

(S)-2-amino-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(287): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(286) (1.0 g, 1.46 mmol) in DMF (5 mL) was added piperidine (1.0 mL)under nitrogen atm at rt. The resultant reaction mixture was stirred atroom temperature for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was washed with pet ether and diethyl ether, dried under vacuumto get 720 mg (crude) of(S)-2-amino-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(287) as a pale brown gum. LC-MS: m z 461.30 [(M+H)⁺]; R_(t): 1.20 min;46.42% purity.

(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(288): To a stirred solution of(S)-2-amino-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(287) (700 mg, 1.52 mmol) in DMF (7 mL) were added(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (79) (516 mg, 1.52 mmol),HATU (867 mg, 2.28 mmol) and DIPEA (0.8 mL, 4.56 mmol) at 0° C. Theresultant reaction mixture was stirred at room temperature for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was quenched with ice water (40mL) and the precipitated solid was filtered, washed with diethyl ether,dried under vacuum to get crude compound. The crude compound waspurified by using column chromatography on silica gel 230-400 mesheluting with 3-5% methanol in dichloromethane to get 900 mg (76% yield)of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(288) as an off-white solid. LC-MS: m z 782.52 [(M+H)⁺]; R_(t): 1.97min; 48.72% purity.

(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(289): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(288) (900 mg, 1.15 mmol) in DMF (5 mL) was added piperidine (0.9 mL)under nitrogen atm at rt. The resultant reaction mixture was stirred atroom temperature for 2 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was washed with pet ether and diethyl ether (50 mL, 7:3 byvolume), dried under vacuum to get 600 mg (93% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(289) as an off-white solid. LC-MS: m z 560.47 [(M+H)⁺]; R_(t): 1.25min; 58.69% purity.

1-azido-N-((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(290): To a stirred solution of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)-5-ureidopentanamide(289) (400 mg, 0.71 mmol) in DMF (5 mL) was added1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (86) (269 mg, 0.71mmol), PyBOP (557 mg, 1.07 mmol) and DIPEA (0.4 mL, 2.14 mmol) at 0° C.The resultant reaction mixture was stirred at room temperature for 2 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was diluted with ethyl acetate,washed with water. The organic layer was dried over anhydrous Na₂SO₄,concentrated under reduced pressure to get 550 mg (84% yield) of1-azido-N-((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(290) as a pale brown gummy. LC-MS: m z 921.62 [(M+H)⁺]; R_(t): 1.65min; 53.50% purity.

1-azido-N-((S)-1-(((S)-1-((4-(2-(hydroxymethyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(291): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(290) (540 mg, 0.59 mmol) in MeOH (6 mL) was added NH₄F (326 mg, 8.79mmol) portion wise at 0° C. and the resultant reaction mixture wasstirred at room temperature for 8 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, the reactionmixture was concentrated under reduced pressure to get crude compound.The crude compound was diluted with water and extracted with 10%methanol in dichloromethane. The combined organic layer was dried overanhydrous Na₂SO₄, filtered and evaporated under reduced pressure toafford 300 mg (crude) of1-azido-N-((S)-1-(((S)-1-((4-(2-(hydroxymethyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(291) as an off-white semi solid. LC-MS: m z 807.57 [(M+H)⁺]; R_(t):1.23 min; 86.16% purity.

(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl(4-nitrophenyl) carbonate (292): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(2-(hydroxymethyl)-1H-imidazol-1-yl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide(291) (150 mg, 0.19 mmol) in DCM (3 mL) was added DIPEA (0.32 mL, 1.86mmol) and the resultant mixture was cooled to 0° C. A solution of4-nitrophenyl carbonochloridate (25) (187 mg, 0.93 mmol) in DCM (2 mL)was added drop wise to the reaction mixture and the reaction mixture wasstirred at 40° C. for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was triturated with diethyl ether to get 250 mg (crude) of(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl(4-nitrophenyl) carbonate (292) as a pale yellow gum. LC-MS: m z 972.50[(M+H)⁺]; R_(t): 1.55 min; 17.10% purity.

(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(293) (TFA salt): To a stirred solution of(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl(4-nitrophenyl) carbonate (292) (250 mg, 0.26 mmol) in NMP (3 mL) wasadded Et₃N (0.36 mL, 2.57 mmol) and the reaction mixture was cooled to0° C.(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (85 mg, 0.16 mmol) was added portion wise and theresultant reaction mixture was stirred at room temperature for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was quenched with ice water. Theprecipitated solid was filtered off, washed with diethyl ether to getthe crude compound. The crude compound was purified by RP-preparativeHPLC to afford 8.8 mg (3% yield) of(1-(4-((23S,26S)-1-azido-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)phenyl)-1H-imidazol-2-yl)methyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(293) (TFA Salt) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ10.26 (s, 1H), 8.19-8.10 (m, 2H), 7.86-7.69 (m, 5H), 7.52-7.31 (m, 4H),6.52 (s, 1H), 5.99 (s, 1H), 5.44-5.20 (m, 8H), 4.41-4.39 (m, 1H), 4.23(t, J=7.2 Hz, 1H), 3.60-3.45 (m, 25H), 3.17-2.94 (m, 4H), 2.39 (s, 7H),2.15-2.08 (m, 2H), 2.00-1.86 (m, 3H), 1.76-1.61 (m, 2H), 1.49-1.33 (m,2H), 0.89-0.82 (m, 9H). LC-MS (method 41): m/z 1268.21 [(M+H)⁺]; R_(t):2.08 min; 95.42% purity, HP-LC (method 41): R_(t): 3.64 min; 96.29%purity.

Example 45: Synthesis of4-((32S,35S)-35-(4-Aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(304)

4-(((Tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyaniline (295): To astirred solution of (4-amino-3-methoxyphenyl)methanol (294) (2.0 g,13.057 mmol) in DMF (15 mL) was added imidazole (1.778 g, 26.113 mmol)followed by tert-butyl(chloro)diphenylsilane (4.306 g, 15.668 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with water (15 mL) andextracted with EtOAc (2×30 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to getcrude compound The crude compound was purified by flash chromatographyeluting with 8% EtOAc in hexane to get 1.6 g (31% yield) of4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyaniline (295) as apale brown gum. LCMS: MH⁺392, retention time 2.61 min.

(9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(296): To a stirred solution ofN₂-(((9H-fluoren-9-yl)methoxy)carbonyl)-N₆-(diphenyl(p-tolyl)methyl)-L-lysine(149) (3.0 g, 4.802 mmol) in DMF (30 mL) was added DIPEA (2.516 mL,14.405 mmol), HATU (3.651 g, 9.603 mmol) and4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxy aniline (295) (1.88g, 4.802 mmol) at 0° C. The resultant reaction mixture was stirred atr.t for 16 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was quenched with icewater. The precipitated solid was filtered off and dried under vacuum toget 2 g (42% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(296) as an off-white solid. LCMS: MH⁺998.3, retention time 5.27 min.

(S)-2-Amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(297): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(296) (2 g, 2.003 mmol) in DMF (15 ml), 30% piperidine in DMF (6 ml) wasadded at rt. The resultant reaction mixture was stirred at r.t for 3 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 1.4 g (90% yield) of(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(297) as a pale brown gum. LCMS: MH⁺776, retention time 4.10 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(298): To a stirred solution of(((N-(9-Fluorenylmethoxycarbonyl)-L-phenylalanine (152) (700 mg, 1.807mmol), in DMF (20 mL) was added DIPEA (0.947 mL, 5.421 mmol), HATU (1.37g, 3.614 mmol) and(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(297) (1.402 g, 1.807 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 1.3 g (63% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(298) as an off-white solid. LCMS: MH⁺1145, retention time 5.22 min.

(S)-2-((S)-2-Amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(299): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate(298) (1.3 g, 1.135 mmol) in DMF (10 mL), 30% piperidine in DMF (4 ml)was added at rt. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 1 g (95% yield) of(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(299) as an off-white solid. LCMS: MH⁺923, retention time 3.90 min.

1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(300): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (550mg, 1.075 mmol) in DMF (10 mL) was added DIPEA (0.563 mL, 3.225 mmol),HATU (817.58 mg, 2.15 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(299) (992.64 mg, 1.075 mmol) at 0° C. The resultant reaction mixturewas stirred at r.t for 6 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×20 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 2% MeOH in DCM to get0.900 g (59% yield) of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(300) as a pale brown gum. LCMS: MH⁺1416, retention time 3.51 min.

1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methoxyphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(301): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-methoxyphenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(300) (1 g, 0.706 mmol) in methanol (20 mL) was added NH₄F (261 mg,7.058 mmol) at rt. The resultant reaction mixture was stirred at r.t for6 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get a crude residue. The residue obtained was diluted withwater (15 mL) and extracted with EtOAc (2×20 mL). The combined organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 5% MeOH in DCM to get 0.600 g (72% yield) of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methoxyphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(301) as an pale brown gum. LCMS: MH⁺1178, retention time 3.74 min.

4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl(4-nitrophenyl) carbonate (302): To a stirred solution of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)-2-methoxyphenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(301) (300 mg, 0.255 mmol) in DCM (10 mL) was added pyridine (0.103 mL,1.274 mmol), 4-nitrophenyl chloroformate (25) (154.04 mg, 0.764 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 2.5% MeOH in DCM to get 0.150 g (44% yield)of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl(4-nitrophenyl) carbonate (302) as a pale brown gum. LCMS: MH⁺1344,retention time 2.57 min.

4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(303): To a stirred solution of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl(4-nitrophenyl) carbonate (302) (220 mg, 0.164 mmol) in NMP (2.5 mL) wasadded TEA (0.068 mL, 0.492 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (71.33 mg, 0.164 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 8h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was quenched with water (15 mL) and extracted with 10% methanolin chloroform (2×20 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. To the crudematerial diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography eluting with 4% MeOH in DCM to get 0.250 g (93% yield) of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(303) as an off-white solid. LCMS: MH⁺1640, retention time 2.47 min.

4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-benzyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(304): To a stirred solution of4-((32S,35S)-1-azido-32-benzyl-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)-3-methoxybenzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(303) (250 mg, 0.152 mmol) in DCM 5 ml 1% TFA in DCM was added at 0° C.and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain the desiredproduct (304) (19 mg, 9% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 9.04 (s, 1H), 8.11-8.13 (d, 2H), 8.02-8.05 (t, 2H),7.76-7.79 (m, 4H), 7.30 (s, 1H), 7.24-7.25 (d, 2H), 7.10-7.21 (m, 5H),6.51 (s, 1H), 5.41 (s, 2H), 5.28 (s, 3H), 5.09 (s, 2H), 4.51-4.52 (m,2H), 3.29-3.80 (m, 44H), 3.03 (s, 1H), 2.76-2.79 (t, 3H), 2.37 (s, 3H),2.28-2.32 (t, 3H), 1.39-2.19 (m, 10H) 0.89-0.82 (t, 9H), LCMS: MH⁺ 1383,retention time 2.07.

Example 46: Synthesis of1-azido-N-((S)-1-(((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(313)

(5-Aminopyridin-2-yl)methanol (306): (5-nitropyridin-2-yl)methanol (305)(500 mg, 3.265 mmol) was taken in par shaker vessel in presence of MeOH(10 ml). Then Pd—C (50 mg) was added to it and kept the reaction mixtureat 40 psi under hydrogen atmosphere for 2h. The progress of the reactionwas monitored by TLC. After completion of starting material, reactionmixture was filtered through sintered funnel. The filtrate wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 1.5% MeOH inDCM to get 0.350 g (86% yield) of (5-aminopyridin-2-yl) methanol (305)as a pale brown gum and was used for the next step.

(9H-Fluoren-9-yl)methyl(S)-(1-((6-(hydroxymethyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(307): (5-aminopyridin-2-yl)methanol (306) (0.76 g, 6.129 mmol),Fmoc-Cit-OH (76) (2.679 g, 6.742 mmol), EEDQ (4.542 g, 13.387 mmol) weremixed in DCM-THF (1:1) (100 ml) and stirred at ambient temperature undernitrogen for 16 h. The reaction mixture was reduced to dryness undervacuum and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (5-10%). The solvent was evaporatedunder vacuum to obtain 1.7 g (55% yield) of (9H-fluoren-9-yl) methyl(S)-(1-((6-(hydroxymethyl) pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (307) as an off-white solid.LCMS: MH⁺504, retention timel.55 min.

(9H-Fluoren-9-yl)methyl(S)-(1-((6-(chloromethyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(308): To a stirred solution of (9H-fluoren-9-yl) methyl(S)-(1-((6-(hydroxymethyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(307) (300 mg, 0.596 mmol) in THF (20 mL), SOCl2 (0.052 ml, 0.715 mmol)was added at OoC. The resultant reaction mixture was stirred at r.t for2 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 2% MeOH in DCM to get 0.200 g (64% yield) of(9H-fluoren-9-yl)methyl(S)-(1-((6-(chloromethyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(308) as an off-white solid. LCMS: MH⁺522, retention time 2.94 min.

(9H-Fluoren-9-yl)methyl((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(309): To a stirred solution of(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (50 mg, 0.094 mmol) in DMF (5 mL) was added K₂CO₃(19.5 mg, 0.141 mmol) at 0° C. and stirred the reaction mixture for 15min. Then (9H-fluoren-9-yl)methyl(S)-(1-((6-(chloromethyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(308) (73.65 mg, 0.141 mmol) and KI (46.84 mg, 0.282 mmol) were added.The resultant reaction mixture was stirred at r.t for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (10 mL) and extractedwith EtOAc (2×10 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound The crude compound was purified by flash chromatography elutingwith 3% MeOH in DCM to get 0.050 g (58% yield) of(9H-fluoren-9-yl)methyl((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(309) as an off-white solid. LCMS: MH⁺921, retention time 3.09 min.

(S)-2-Amino-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(310): A solution of (9H-fluoren-9-yl)methyl((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(309) (230 mg, 0.25 mmol) in DMF (5 ml) was treated with piperidine(0.074 ml, 0.749 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.085 g (49% yield) of(S)-2-amino-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(310) crude as a sticky liquid. LCMS: MH⁺699, retention time 2.51 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(311): To a stirred solution of Fmoc-L-Valine (79) (49.542 mg, 0.146mmol) in DMF (5 mL) was added DIPEA (0.05 mL, 0.304 mmol), HATU (92.50mg, 0.243 mmol) and(S)-2-amino-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(310) (85 mg, 0.122 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with 3.5% MeOH inDCM to get 0.120 g (96% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(311) as a sticky solid. LCMS: MH⁺1020, retention time 3.24 min.

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(312): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(311) (120 mg, 0.118 mmol) in DMF (2 ml) was treated with piperidine(0.035 ml, 0.353 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.093 g (99% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(312) crude as a sticky liquid. LCMS: MH⁺798, retention time 2.69 min.

1-azido-N-((S)-1-(((S)-1-((6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(313): To a stirred solution of Azido-PEG9-acid (84.623 mg, 0.165 mmol)in DMF (3 mL) was added DIPEA (0.06 mL, 0.345 mmol), HATU (104.82 mg,0.276 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)methyl)pyridin-3-yl)-5-ureidopentanamide(312) (110 mg, 0.138 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to isolate the desired product (313) (14 mg, 8%yield) as a brown solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.17 (s, 1H),8.70-8.71 (d, 1H), 8.15-8.16 (d, 1H), 8.03-8.05 (d, 1H), 7.85-7.87 (d,1H), 7.73-7.76 (d, 1H), 7.45-7.47 (d, 1H), 7.30 (s, 1H), 6.50 (s, 1H),5.98 (s, 1H), 5.27-5.52 (m, 6H), 4.39 (s, 1H), 4.21-4.27 (m, 3H), 3.97(s, 2H), 3.36-3.59 (m, 26H), 2.76-3.04 (m, 5H), 2.27-2.2.38 (m, 4H),1.39-1.99 (m, 12H), 0.82-0.89 (m, 9H). LCMS: MH⁺1291, retention time2.18 min.

Example 47: Synthesis of1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaoctadecan-18-amide(318)

(9H-Fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(314): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (1.25 g, 3.186 mmol) in DMF (15 mL) was added NaH (0.115 g, 4.778mmol) at 0° C. and stirred the reaction mixture for 15 min. Then(9H-fluoren-9-yl) methyl (S)-(1-((4-(iodomethyl) phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (227) (3.90 g, 6.371 mmol)was added. The resultant reaction mixture was stirred at r.t for 1 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with water (10 mL),neutralized with acetic acid and extracted with DCM (2×10 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 2.5% MeOH in DCM to get1.25 g (45% yield) of (9H-fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(314) as an off-white solid. LCMS: MH⁺877.9, retention time 1.93 min.

(S)-2-Amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(315): A solution of (9H-fluoren-9-yl)methyl((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(314) (1.25 g, 1.427 mmol) in DMF (10 ml) was treated with piperidine(0.429 ml, 4.281 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.800 g (86% yield) of(S)-2-amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(315) crude as a sticky liquid. LCMS: MH⁺655.5, retention time 1.54 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(316): To a stirred solution of Fmoc-L-Valine (79) (497.64 mg, 1.466mmol) in DMF (10 mL) was added DIPEA (0.533 mL, 3.055 mmol), HATU (0.929mg, 2.444 mmol) and(S)-2-amino-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(315) (800 mg, 1.222 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by flash chromatography eluting with (3-4)% MeOHin DCM to get 1 g (84% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(316) as a sticky solid. LCMS: MH⁺977.4, retention time 2.71 min.

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(317): A solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(316) (500 mg, 0.26 mmol) in DMF (7 ml) was treated with piperidine(0.154 ml, 1.537 mmol) and the reaction mixture stirred at 0° C. undernitrogen atmosphere for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material the reaction mixture wasreduced to dryness under vacuum to get 0.400 g (quantitative yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(317) crude as a sticky liquid and this material was used for the nextstep without further purification. LCMS: MH⁺754.3, retention time 2.74min.

1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaoctadecan-18-amide(318): To a stirred solution of Azido-PEG5-acid (84) (80.07 mg, 0.239mmol) in DMF (5 mL) was added DIPEA (0.087 mL, 0.497 mmol), HATU (151.31mg, 0.398 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(317) (150 mg, 0.199 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired product (318) (12 mg, 6%yield) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.93 (s, 1H),8.07-8.09 (d, 1H), 7.98-8.00 (s, 1H), 7.71-7.73 (d, 2H), 7.55-7.60 (d,2H), 7.48-7.50 (d, 2H), 7.27 (s, 1H), 6.48 (s, 1H), 5.97 (s, 1H),5.39-5.42 (d, 4H), 5.30 (s, 4H), 4.38 (s, 1H), 4.14-4.18 (t, 1H),2.94-3.59 (m, 23H), 2.32-2.39 (m, 2H), 1.23-1.94 (m, 13H), 0.85 (s, 9H).LCMS: MH⁺1071, retention time 2.91 min.

Example 48: Synthesis of1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(319)

1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(319): To a stirred solution of Azido-PEG9-acid (86) (162.86 mg, 0.318mmol) in DMF (5 mL) was added DIPEA (0.116 mL, 0.663 mmol), HATU (0.202mg, 0.531 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(317) (200 mg, 0.265 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired product (319) (55 mg, 17%yield) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.91 (s, 1H),8.63 (s, 1H), 8.40 (s, 1H), 8.07-8.09 (d, 1H), 7.71 (s, 2H), 7.48-7.60(m, 4H), 7.27 (s, 1H), 6.48 (s, 1H), 5.95 (s, 1H), 5.38-5.42 (d, 4H),5.30 (s, 4H), 4.38 (s, 1H), 4.16 (s, 1H), 2.96-3.58 (m, 39H), 1.25-1.86(m, 15H), 0.87 (s, 9H). LCMS: MH⁺1247.8, retention time 2.46 min.

Example 49: Synthesis of1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(320)

1-azido-N-((S)-1-(((S)-1-((4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(320): To a stirred solution of Azido-PEG1I-acid (88) (190.91 mg, 0.318mmol) in DMF (5 mL) was added DIPEA (0.116 mL, 0.663 mmol), HATU (0.202mg, 0.531 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)methyl)phenyl)-5-ureidopentanamide(317) (200 mg, 0.265 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was initially purified by flash chromatography, finallypurified by RP-prep-HPLC to obtain the desired product (320) wasisolated (45 mg, 13% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆):δ 9.90 (s, 1H), 8.07-8.09 (d, 1H), 7.97-7.99 (s, 1H), 7.71-7.73 (d, 2H),7.75-7.60 (t, 2H), 7.48-7.50 (d, 2H), 7.27 (s, 1H), 5.38-5.42 (d, 1H),5.30 (s, 4H), 4.38 (s, 4H), 4.14-4.16 (s, 1H), 2.95-3.59 (m, 51H),2.32-2.39 (m, 2H), 1.26-1.92 (m, 10H), 0.87 (s, 9H). LCMS: MH⁺1335.8,retention time 2.46 min.

Example 50: Synthesis of1-azido-N-((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(321)

1-azido-N-((S)-1-(((R)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(321): To a stirred solution of azido peg-11-acid (88) (83.22 mg, 0.139mmol) in DMF (5 mL) was added DIPEA (0.05 mL, 0.289 mmol), HATU (87.944mg, 0.231 mmol) and(R)-2-((S)-2-amino-3-methylbutanamido)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-5-ureidopentanamide(317) (80 mg, 0.166 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (10 mL) and extracted with EtOAc (2×10 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM. Thesolvent was evaporated under vacuum and the material was finallypurified by RP prep-HPLC to obtain the desired product (321) (20 mg,14%) as a sticky solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.44-8.46 (d, 1H),8.26-8.28 (d, 1H), 7.91-7.93 (d, 1H), 7.76-7.79 (d, 1H), 7.31 (s, 1H),6.50 (s, 1H), 5.93 (s, 1H), 5.55 (s, 1H), 5.42 (m, 4H), 5.16 (s, 2H),4.23 (s, 1H), 2.86-3.86 (m, 41H), 1.24-2.33 (m, 23H), 0.86-0.90 (t, 3H),0.80-0.83 (t, 6H). LCMS: MH⁺1273.5, retention time 2.41 min.

Example 51: Synthesis of4-((S)-6-amino-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(325)

7-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)heptanamide(322): To a stirred solution of 7-azidoheptanoic acid (701.70 mg, 1.07mmol) in DMF (10 mL) was added DIPEA (0.46 mL, 2.68 mmol), HATU (817.31mg, 2.15 mmol) and(S)-2-((S)-2-amino-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)-N-(4-(hydroxymethyl)phenyl)hexanamide(218) (184 mg, 1.07 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 1.5% MeOH in DCM to get0.33 g (38% yield) of7-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)heptanamide(322) as a pale brown gum. LCMS: MH⁺808, retention time 2.23 min.

4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(4-nitrophenyl) carbonate (323): To a stirred solution of7-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)heptanamide(322) (190 mg, 0.23 mmol) in DCM (15 mL) was added TEA (0.13 mL, 0.94mmol), 4-nitrophenyl chloroformate (25) (94.90 mg, 0.47 mmol) at 0° C.The resultant reaction mixture was stirred at r.t for 6 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by flashchromatography eluting with 2% MeOH in DCM to get 0.09 g (39% yield) of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(4-nitrophenyl) carbonate (323) as a pale brown gum. LCMS: MH⁺973,retention time 4.35 min.

4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(324): To a stirred solution of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl(4-nitrophenyl) carbonate (323) (210 mg, 0.216 mmol) in NMP (5 mL) wasadded TEA (0.07 mL, 0.53 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (114.71 mg, 0.216 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 16h. The progress of thereaction was monitored by LCMS. After completion of starting material,reaction mixture was quenched with water (15 mL) and extracted with 10%methanol in chloroform (2×20 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. To thecrude material diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography eluting with 5% MeOH in DCM to get 0.170 g (62% yield) of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(324) as an off-white solid. LCMS: MH⁺1269, retention time 2.52 min.

4-((S)-6-amino-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(325): To a stirred solution of4-((S)-2-((S)-2-(7-azidoheptanamido)-3-phenylpropanamido)-6-((diphenyl(p-tolyl)methyl)amino)hexanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(324) (170 mg, 0.134 mmol) in DCM 5 ml 1% TFA in DCM was added at 0° C.and the resultant reaction mixture was stirred at r.t for 1 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was triturated withdiethyl ether and finally purified by RP-prep-HPLC to obtain the desiredproduct (325) (40 mg, 30% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): (10.03 (s, 1H), 8.20-8.22 (d, 1H), 8.00-8.02 (d, 2H),7.76-7.79 (d, 1H), 7.60-7.62 (m, 4H), 7.36-7.38 (d, 2H), 7.31 (s, 1H),7.19-7.26 (m, 3H), 7.14 (s, 1H), 7.08 (s, 1H), 5.43 (s, 2H), 5.28 (s,3H), 5.08 (s, 2H), 4.50-4.55 (m, 2H), 3.24-3.3.36 (m, 5H), 1.86-2.77 (m,15H), 1.08-1.54 (m, 14H), 0.85-0.89 (t, 3H). LCMS: MH⁺1014, retentiontime 1.65 and 1.62 min.

Example 52: Synthesis of4-((32S,35S)-1-azido-32-isopropyl-40-methyl-30,33,36,39-tetraoxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37,40-tetraazadotetracontan-42-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(448)

tert-butyl(S)-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoyl)glycinate(436): tert-butyl glycinate (435) (2.53 g, 15.098 mmol), Fmoc-Cit-OH(76) (5 g, 12.582 mmol), EEDQ (9.334 g, 37.745 mmol) were mixed inDCM-THF (1:1) (400 ml) and stirred at ambient temperature under nitrogenfor 16 h. The reaction mixture was reduced to dryness under vacuum andpurified by column chromatography on a silica cartridge eluting withmethanol/DCM gradient (3-10%). The solvent was evaporated under vacuumto obtain 3.5 g (54% yield) of tert-butyl (S)-(2-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)-5-ureidopentanoyl)glycinate (436) as a whitesolid. LCMS: MH⁺511, retention time 3.14 min.

(S)-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoyl)glycine(437): To a stirred solution of tert-butyl (S)-(2-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)-5-ureidopentanoyl)glycinate (436) (3.5 g,6.863 mmol) in DCM (20 mL) was added TFA 5 ml at 0° C. The resultantreaction mixture was stirred at r.t for 16 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was concentrated to get the crude compound. The crudematerial was triturated with diethyl ether to obtained 3 g (96% yield)of (S)-(2-((((9H-fluoren-9-yl) methoxy) carbonyl)amino)-5-ureidopentanoyl)glycine (437) as a white solid. LCMS: MH⁺454,retention time 2.61 min

4-(((Tert-butyldiphenylsilyl)oxy)methyl)aniline (159): To a stirredsolution of (4-aminophenyl)methanol (75) (3.0 g, 24.39 mmol) in DMF (15mL) was added imidazole (3.31 g, 48.78 mmol) followed bytert-butyl(chloro)diphenylsilane (7.35 g, 26.82 mmol) at 0° C. Theresultant reaction mixture was stirred at r.t for 16 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (15 mL) and extractedwith EtOAc (2×100 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound The crude compound was purified by flash chromatography elutingwith 10% EtOAc in hexane to get 3.5 g (40% yield) of4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159) as a pale browngum. LCMS: MH⁺362, retention time 1.25 min.

(9H-Fluoren-9-yl)methyl(2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)carbamate(439): To a stirred solution ofN-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (438) (1.5 g,4.819 mmol) in DMF (15 mL) was added DIPEA (2.525 mL, 14.456 mmol), HATU(3.664 g, 9.637 mmol) and4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159) (1.742 g, 4.819mmol) at 0° C. The resultant reaction mixture was stirred at r.t for 6h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was quenched with ice water. Theprecipitated solid was filtered off and dried under vacuum to get 2.5 g(79% yield) of (9H-fluoren-9-yl)methyl(2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)carbamate(439) as an off-white solid. LCMS: MH⁺655, retention time 4.90 min.

N-(4-(((Tert-butyldiphenylsilyl)oxy)methyl)phenyl)-2-(methylamino)acetamide(440): To a stirred solution of (9H-fluoren-9-yl)methyl(2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)carbamate(439) (3 g, 4.585 mmol) in DMF (15 ml), 30% piperidine in DMF (7 ml) wasadded at rt. The resultant reaction mixture was stirred at r.t for 3 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 1.5 g (76% yield) ofN-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-2-(methylamino)acetamide (440) as a brown liquid.LCMS: MH⁺433, retention time 2.14 min.

(9H-Fluoren-9-yl)methyl(S)-(1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(441): To a stirred solution of(S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoyl)glycine(437) (3.5 g, 7.701 mmol), in DMF (20 mL) was added DIPEA (4.037 mL,23.103 mmol), HATU (5.856 g, 15.402 mmol) andN-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-2-(methylamino)acetamide (440) (3.332 g, 7.701 mmol)at 0° C. The resultant reaction mixture was stirred at r.t for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with ice water. Theprecipitated solid was filtered off and dried under vacuum to get 4 g(60% yield) of (9H-fluoren-9-yl)methyl(S)-(1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(441) as an off-white solid. LCMS: MH⁺869, retention time 2.16 min.

(S)-2-Amino-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(442): To a stirred solution of (9H-fluoren-9-yl)methyl(S)-(1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate(441) (4 g, 4.603 mmol) in DMF (15 mL), 30% piperidine in DMF (12 ml)was added at rt. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 100% EtOAc to get 2.1 g (70% yield) of(S)-2-amino-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(442) as an off-white solid. LCMS: MH⁺647, retention time 1.80 min.

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(443): To a stirred solution of Fmoc-L-Valine (1.103 g, 3.249 mmol), inDMF (20 mL) was added DIPEA (1.687 mL, 9.747 mmol), HATU (2.471 g, 6.498mmol)(S)-2-amino-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(442) (2.1 g, 3.249 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with ice water. The precipitated solid was filtered off anddried under vacuum to get 2.6 g (82% yield) of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(443) as an off-white solid. LCMS: MH⁺968, retention time 2.20 min.

(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(444): To a stirred solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(443) (2.6 g, 2.685 mmol) in DMF (10 mL), 30% piperidine in DMF (9 ml)was added at rt. The resultant reaction mixture was stirred at r.t for 2h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 2% MeOH in DCM to get 1.9 g (94% yield) of(S)-2-((S)-2-amino-3-methylbutanamido)-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(444) as a sticky solid. LCMS: MH⁺746, retention time 1.64 min.

1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(445): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (1 g,1.957 mmol) in DMF (8 mL) was added DIPEA (1.023 mL, 5.871 mmol), HATU(1.487 g, 3.914 mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)-5-ureidopentanamide(444) (1.46 g, 1.957 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with EtOAc (2×30 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 3% MeOH in DCM to get 2.1g (86% yield) of1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(445) as a pale brown gum. LCMS: MH⁺1239, retention time 1.80 min.

1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(446): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(445) (1.4 g, 1.129 mmol) in methanol (15 mL) was added NH₄F (461 mg,13.552 mmol) at rt. The resultant reaction mixture was stirred at r.tfor 6 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get a crude residue. The residue obtained wasdiluted with water (15 mL) and extracted with EtOAc (2×20 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound. The crude compound waspurified by flash chromatography eluting with 5% MeOH in DCM to get0.800 g (71% yield) of1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(446) as an pale brown gum. LCMS: MH⁺1001, retention time 1.87 min.

4-((32S,35S)-1-Azido-32-isopropyl-40-methyl-30,33,36,39-tetraoxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37,40-tetraazadotetracontan-42-amido)benzyl(4-nitrophenyl) carbonate (447): To a stirred solution of1-azido-N-((S)-1-(((S)-1-((2-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)(methyl)amino)-2-oxoethyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(446) (600 mg, 0.60 mmol) in DCM (10 mL) was added TEA (0.418 mL, 2.998mmol), and 4-nitrophenyl chloroformate (25) (362.612 mg, 1.799 mmol) at0° C. The resultant reaction mixture was stirred at r.t for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 2.5% MeOH in DCM to get 0.300 g (42% yield)of4-((32S,35S)-1-azido-32-isopropyl-40-methyl-30,33,36,39-tetraoxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37,40-tetraazadotetracontan-42-amido)benzyl(4-nitrophenyl) carbonate (447) as a pale brown gum. LCMS: MH⁺1166,retention time 0.90 min.

4-((32S,35S)-1-Azido-32-isopropyl-40-methyl-30,33,36,39-tetraoxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37,40-tetraazadotetracontan-42-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(448): To a stirred solution4-((32S,35S)-1-azido-32-isopropyl-40-methyl-30,33,36,39-tetraoxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37,40-tetraazadotetracontan-42-amido)benzyl(4-nitrophenyl) carbonate (447) (300 mg, 0.257 mmol) in NMP (2 mL) wasadded TEA (0.105 ML, 0.771 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (111.81 mg, 0.257 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 8h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was quenched with water (15 mL) and extracted with 10% methanolin chloroform (2×20 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. To the crudematerial diethylether was added and solid precipitated out. It wasfiltered and the solid material was passed through Combi-flash columnchromatography and finally purified by RP-prep HPLC to obtain thedesired product (449) (129 mg, 34% yield) as an off-white sticky solid.¹H NMR (400 MHz, DMSO-d₆): (10.01 (s, 1H), 10.03 (s, 1H), 8.03-8.06 (d,1H), 7.95-7.97 (d, 1H), 7.87-7.90 (m, 1H), 7.81-7.83 (d, 1H), 7.76-7.79(d, 1H), 7.56-7.61 (t, 2H), 7.35-7.39 (t, 2H), 7.31 (s, 1H), 6.50 (s,1H), 5.44 (s, 1H), 5.28 (s, 1H), 5.07 (s, 2H), 4.31 (s, 4H), 4.12-4.30 9m, 5H), 2.50-3.98 (m, 28H), 1.36-2.42 (m, 21H), 0.79-0.89 (m, 13H).LCMS: MH⁺1462, retention time 2.16 min.

Example 53: Synthesis of4-((S)-1-((32S,35S)-1-azido-32-isopropyl-30,33,36-trioxo-35-(3-ureidopropyl)-3,6,9,12,15,18,21,24,27-nonaoxa-31,34,37-triazanonatriacontan-39-oyl)pyrrolidine-2-carboxamido)benzyl((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(464)

Compound (464) can be prepared by using the methods disclosed herein.

Example 54: Synthesis of(S)-1-azido-N-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)disulfanyl)-2-methylpropyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide(332)

(S)-9-(3-Bromopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(326): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (2 gm, 5.102 mmol) in DMSO (4 ml), K₂CO₃ (7 gm, 51.02 mmol)wasadded to the reaction mixture at rt, after 5 mins 1,3 Dibromo propane(12.36 g, 61.224 mmol)was added and the reaction mixture was stirred for5h at rt, under Nitrogen atmosphere. The progress of reaction wasmonitored by TLC, after full conversion of starting material, ether wasadded to the reaction mixture, and the resulting solid precipitate wasfiltered and purified by column chromatography on a silica cartridgeeluting with methanol/DCM gradient (5-10%). The solvent was evaporatedunder vacuum to obtain desired product 1.3 g (49% yield) of(S)-9-(3-bromopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(326) as a yellow solid. LCMS: MH⁺514, retention time 1.87 min.

(S)—S-(3-((4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)ethanethioate (327): A solution of(S)-9-(3-bromopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(326) (1.3 g, 0.292 mmol) in acetone (30 ml) was treated with potassiumthioacetate (430 mg 0.438 mmol) and the reaction mixture refluxed at 78°C. under nitrogen atmosphere for 30 mins. The progress of reaction wasmonitored by LCMS, after full conversion of starting material thereaction mixture was reduced to dryness under vacuum and purified bycolumn chromatography on a silica cartridge eluting with 2% Methanol inDCM. The solvent was evaporated under vacuum to obtain desired product0.65 g (50% yield) of(S)—S-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)ethanethioate (327) as an off-white solid. LCMS: MH⁺509, retention time1.86 min.

(S)-4-Ethyl-4-hydroxy-9-(3-(pyridin-2-yldisulfanyl)propoxy)-12,14-dihydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-3(4H)-one(329): To a stirred solution of(S)—S-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)ethanethioate (327) (0.650 g, 0.098 mmol), in methanol (30 ml) was addedPyridine bi sulphide (328) (0.364 g, 0.128 mmol) and degassed with N₂gas after 15 mins sodium methoxide (0.689 g, 0.98 mmol) was added at rt,and stirred at room temperature under nitrogen atmosphere for 6 h. Thereaction mixture was reduced to dryness under vacuum and purified bycolumn chromatography on a silica cartridge eluting with (5-10)%methanol in DCM. The solvent was evaporated under vacuum to obtaindesired product 0.26 g (36% yield) of(S)-4-ethyl-4-hydroxy-9-(3-(pyridin-2-yldisulfanyl)propoxy)-12,14-dihydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-3(4H)-one (329) as a whitesolid. LCMS: MH⁺577, retention time 1.91 min.

(S)-9-(3-((1-Amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331):(S)-4-ethyl-4-hydroxy-9-(3-(pyridin-2-yldisulfanyl)propoxy)-12,14-dihydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-3(4H)-one(329) (0.260 g, 0.435 mmol), 1-amino-2-methylpropane-2-thiolhydrochloride (330) (0.093 g, 0.435 mmol) were taken in methanol (25 ml)and was stirred at room temperature under nitrogen atmosphere for 3h.The solvent was reduced to dryness under vacuum and purified by columnchromatography on a silica cartridge eluting with (5-10) % methanol inDCM. The solvent was evaporated under vacuum to obtain desired product0.08 g (33% yield) of(S)-9-(3-((1-amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331) as oily liquid. LCMS: MH⁺570, retention time 2.90 min.

(S)-1-Azido-N-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)disulfanyl)-2-methylpropyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide(332):(S)-9-(3-((1-amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331) (0.08 g, 0.11 mmol) HATU (80 mg, 0.17 mmol), azido-peg5-acid(55.75 mg, 0.139 mmol) and DIPEA (0.03 ml, 0.15 mmol) were mixed in DMF(0.3 ml) and stirred at ambient temperature under nitrogen atmospherefor 6 h. The reaction mixture was reduced to dryness under vacuum andpurified by column chromatography on a silica cartridge eluting withmethanol/DCM gradient (1-2%). The solvent was evaporated under vacuumand the material was finally purified by RP prep-HPLC to obtain thedesired product (332) as an off-white solid (8 mg, 15% yield). ¹H NMR(400 MHz, DMSO-d₆) δ 8.1 (d, 1H), 7.91 (t, 1H), 7.5 (d, 2H), 7.27 (s,1H), 6.49 (s, 1H), 5.4-5.3 (s, 4H), 4.3 (t, 2H), 3.5 (m, 3H), 3.52 (m,11H), 3.37-3.27 (m, 4H), 2.93 (t, 2H), 2.6 (s, 1H), 2.5 (s, 1H), 2.17(s, 1H), 1.7 (m, 1H),1.33 (t, 3H), 1.29 (m, 6H), 0.86 (t, 3H). LCMS:MH⁺887, retention time 1.76 min.

Example 55: Synthesis of(S)-1-Azido-N-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)disulfanyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(333)

(S)-9-(3-((1-amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331) (0.120 g, 0.211 mmol) HATU (120.053 mg, 0.316 mmol),azido-peg9-acid (86) (129.328 mg, 0.253 mmol) and diisopropylethylamine(81.509 mg, 0.632 mmol) were mixed in DMF (5 ml) and stirred at ambienttemperature under nitrogen atmosphere for 6 h. The reaction mixture wasreduced to dryness under vacuum and purified by column chromatography ona silica cartridge eluting with methanol/DCM gradient (1-2%). Thesolvent was evaporated under vacuum and the material was finallypurified by RP prep-HPLC to obtain the desired product (333) as anoff-white solid (36 mg, 16% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (d,1H), 7.8 (t, 1H), 7.4 (t, 2H), 7.27 (s, 1H), 6.49 (s, 1H), 5.4-5.3 (s,4H), 4.3 (s, 2H), 3.57-3.16 (m, 31H), 2.9 (t, 2H), 2.35 (m, 2H), 2.15(t, 2H), 1.85 (m, 2H), 1.30 (m, 3H), 1.18 (m, 6H), 0.86 (t, 3H), LCMS:MH⁺1063, retention time 2.76 min.

Example 56: Synthesis of(S)-1-azido-N-(2-((3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)disulfanyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amide(334)

(S)-9-(3-((1-amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331) (0.120 g, 0.211 mmol) HATU (120.053 mg, 0.316 mmol),azido-peg11-acid (88) (151.565 mg, 0.253 mmol) and DIPEA (0.104, 0.632mmol) were mixed in DMF (5 ml) and stirred at ambient temperature undernitrogen atmosphere for 6 h. chromatography on a silica cartridgeeluting with methanol/DCM gradient (2%). The solvent was evaporatedunder vacuum and the material was finally purified by RP prep-HPLC toobtain one peak for desired product (334) as an off-white solid (34 mg,14% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (d, 1H), 7.8 (t, 1H), 7.5(d, 2H), 7.27 (s, 1H), 6.49 (s, 1H), 5.4-5.3 (s, 4H), 4.3 (t, 2H),3.6-3.19 (m, 43H), 2.9 (t, 2H), 2.35 (m, 2H), 2.17 (m, 2H), 1.85 (m,2H), 1.30 (m, 3H), 1.14 (m, 6H), 0.86 (t, 3H), LCMS: MH⁺1151, retentiontime 2.76 min.

Example 57: Synthesis of37-azido-8-oxo-11,14,17,20,23,26,29,32,35-nonaoxa-3,4-dithia-7-azaheptatriacontyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(342)

2-(pyridin-2-yldisulfanyl) ethan-1-ol (336): To a stirred solution of2-mercaptoethan-1-ol (335) (0.21 ml, 2.99 mmol) in MeOH (15 mL) wasadded 1,2-di(pyridin-2-yl)disulfane (2) (2 g, 9.08 mmol) and thereaction mixture was degassed with argon for 15 min. The resultantreaction mixture was stirred at r.t for 6 h. The progress of thereaction was monitored by TLC. After completion of starting material,the reaction mixture was concentrated under reduced pressure to getcrude compound. The crude compound was purified by flash chromatographyeluting with 50% EtOAc in hexane to get 1.2 g (71% yield) of2-(pyridin-2-yldisulfanyl) ethan-1-ol (336) as a light yellow gum. ¹HNMR (400 MHz, DMSO-d₆): δ 8.44-8.45 (d, 1H), 7.79-7.85 (m, 2H),7.22-7.25 (m, 1H), 4.97-65.00 (t, 1H), 3.59-3.64 (m, 2H), 2.90-2.93 (t,2H). LCMS: MH⁺188, retention time 1.50 min.

4-nitrophenyl (2-(pyridin-2-yldisulfanyl) ethyl) carbonate (337): To astirred solution of 2-(pyridin-2-yldisulfanyl)ethan-1-ol (336) (1.4 g,7.47 mmol) in DCM (20 mL) was added pyridine (2.41 mL, 29.90 mmol),4-nitrophenyl chloroformate (25) (4.52 g, 22.42 mmol) at rt. Theresultant reaction mixture was stirred at r.t for 2 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by flashchromatography eluting with 30% ethylacetate in hex to get 1.8 g (68%yield) of 4-nitrophenyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate(337) as a pale brown gum. LCMS: MH⁺353, retention time 3.45 min.

2-(pyridin-2-yldisulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(338): To a stirred solution 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl) carbonate (337) (75 mg, 0.21 mmol) inDMSO (1.5 mL) was added TEA (0.07 mL, 0.53 mmol), and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (113 mg, 0.21 mmol) at 0° C. The resultantreaction mixture was stirred at r.t for 3h. The progress of the reactionwas monitored by LCMS. After completion of starting material, reactionmixture was quenched with water (15 mL) and extracted with 10% methanolin DCM (2×10 mL). The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. To the crude materialdiethylether was added and solid precipitated out. It was filtered andthe solid material was passed through Combi-flash column chromatographyeluting with 5% MeOH in DCM to get 0.100 g (72% yield) of2-(pyridin-2-yldisulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(338) as an off-white solid. LCMS: MH⁺649, retention time 3.29 min.

2-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(340): To a stirred solution of tert-butyl (2-mercaptoethyl)carbamate(339) (245.55 mg, 1.38 mmol) in MeOH (50 ml) was added2-(pyridin-2-yldisulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(338) (600 mg, 0.92 mmol) and the reaction mixture was degassed withargon for 15 min. The resultant reaction mixture was stirred at r.t for6 h. The progress of the reaction was monitored by TLC. After completionof starting material, the reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 50% EtOAc in hexane to get 0.400 g(61% yield) of 2-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(340) as a light yellow gum. LCMS: MH⁺715, retention time 3.42 min.

2-((2-aminoethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(341): To a stirred solution of2-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(340) (670 mg, 0.93 mmol) in DCM 5 ml TFA (1 ml) was added at 0° C. andthe resultant reaction mixture was stirred at r.t for 1 h. The progressof the reaction was monitored by LCMS. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was triturated with diethyl etherto obtain 0.500 g (87% yield) of 2-((2-aminoethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(341) as an off-white solid. LCMS: MH⁺615, retention time 2.83 min.

37-azido-8-oxo-11,14,17,20,23,26,29,32,35-nonaoxa-3,4-dithia-7-azaheptatriacontyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(342): To a stirred solution of 2-((2-aminoethyl)disulfanyl)ethyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(341) (500 mg, 0.81 mmol), in DMF (10 mL) was added DIPEA (0.35 mL, 2.03mmol), HATU (618 mg, 1.62 mmol) and azido-peg-9acid (86) (499.32 mg,0.97 mmol) at 0° C. The resultant reaction mixture was stirred at r.tfor 16 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. It was purified by normal columnchromatography followed by RP-prep-HPLC to obtain the desired product(342) (215 mg). ¹H NMR (400 MHz, DMSO-d₆): δ 8.01-8.02 (m, 2H),7.76-7.79 (d, 1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.42 (s, 2H), 5.25 (s,3H), 4.30-4.31 (m, 2H), 3.44-3.60 (m, 37H), 3.37-3.39 (m, 4H), 3.01-3.40(m, 4H), 2.77-2.80 (t, 2H), 2.26-2.50 (m, 6H), 1.84-1.88 (t, 2H),0.85-0.88 (t, 3H). LCMS: MH⁺1108, retention time 3.13 and 3.18 min.

Example 58: Synthesis of4-((1-azido-33-methyl-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatetratriacontan-33-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(352)

4-(acetylthio)benzoic acid (344): To a stirred solution of4-Mercaptobenzoic acid (343) (3.0 g, 19.48 mmol) in acetic anhydride(8.94 g, 87 mmol) was added pyridine (4.61 g, 58.4 mmol) at r. Theresultant reaction mixture was stirred at r.t for 4 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (15 mL) and extractedwith EtOAc (2×30 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound The crude compound was purified by flash chromatography elutingwith 20% EtOAc in hexane to get 2.4 g (70% yield) of4-(acetylthio)benzoic acid (344) as white solid. LCMS: MH⁺197, retentiontime 1.62 min

(S-(4-(Hydroxymethyl)phenyl) ethanethioate (345): To a stirred solution4-(acetylthio)benzoic acid (344) (2.4 g, mmol) in THF (25 ml) was addedBH₃-THF (15 ml), at (−0.10° C.). The resultant reaction mixture wasstirred at r.t for 6 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasquenched with 1N HCl and extracted by ethyl acetate (2×20 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get crude compound The crude compound waspurified by flash chromatography eluting with 40% EtOAc in hexane to get2 g (75% yield) of (S-(4-(hydroxymethyl)phenyl) ethanethioate (345). ¹HNMR (400 MHz, DMSO-d₆): δ 7.4-7.1 (d, 4H), 5.3 (t, 1H), 4.4 (d, 2H) 2.4(s, 3H).

(4-(Pyridin-2-yldisulfanyl)phenyl)methanol (346): To a stirred solutionof (S-(4-(hydroxymethyl)phenyl) ethanethioate (345) (2 g, 10.9 mmol) inmethanol (50 ml), sodium methoxide (0.593 g 10.9 mmol) followed bypyridine bi sulphide (328) (2.417 g 10.9 mmol) was added at rt under N₂atmosphere. The resultant reaction mixture was stirred at r.t for 3 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 50% EtOAc/hexane to get 1.3 g (42% yield) of(4-(pyridin-2-yldisulfanyl) phenyl) methanol (346) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.4 (S, 1H). 7.8-7.2 (m, 7H) 5.2 (t,1H), 4.4 (d, 2H),

Tert-butyl (2-mercapto-2-methylpropyl)carbamate (347): To a stirredsolution of 1-amino-2-methylpropane-2-thiol hydrochloride (330) (2 g,19.23 mmol), in water (20 mL) was added boc anhydride (8.38 g, 38.46mmol), in ACN (20 ml) and sodium bicarbonate (3.2 g, 38 mmol) in 5 mlwater at r.t. The resultant reaction mixture was stirred at r.t for 23h. The progress of the reaction was monitored by TLC, a non polar KMO4active spot was generated. After completion of starting material,reaction mixture was quenched with water and extracted by ethyl acetate(2×20 ml). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to get crude compound The crudecompound was purified by flash chromatography eluting with 10% EtOAc inhexane to get 1.3 g (30% yield) of Tert-butyl(2-mercapto-2-methylpropyl)carbamate (347) as an off-white solid. ¹H NMR(400 MHz, DMSO-d₆): δ 7.5-7.2 (d, 4H), 6.9 (t, 1H), 5.2 (t, 1H), 4.4 (d,2H), 3.0 (d, 2H), 1.38 (s 9H), 1.15 (s, 6H),

Tert-butyl(2-((4-(Hydroxymethyl)phenyl)disulfanyl)-2-methylpropyl)carbamate (348):To a stirred solution of Tert-butyl (2-mercapto-2-methylpropyl)carbamate(347) (0 0.5 g, 2 mmol) in methanol (30 mL),(4-(pyridin-2-yldisulfanyl)phenyl)methanol (346) (0.951 g, 3 mmol) inmethanol (15 ml) was added at r.t and resultant mixture degassed bynitrogen for 15 mins. Then reaction mixture was stirred at r.t for 3 hunder N₂ atmosphere. The progress of the reaction was monitored by TLC.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound waspurified by flash chromatography eluting with 60% EtOAc/hexane to get0.40 g (57% yield) of Tert-butyl(2-((4-(hydroxymethyl)phenyl)disulfanyl)-2-methylpropyl)carbamate (348)as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.5 (d, 2H), 7.3 (d,2H), 6.9 (t, 1H), 5.2 (t, 1H), 4.4 (d, 2H), 3.2 (d, 2H), 1.3 (s 9H),1.14 (s, 6H), LCMS: MH⁺343, retention time 3.41 min.

Tert-buty(2-methyl-2-((4-((((4-Nitrophenoxy)carbonyl)oxy)methyl) phenyl)disulfanyl) propyl)carbamate (349): To a stir solution of Tert-butyl(2-((4-(hydroxymethyl)phenyl)disulfanyl)-2-methylpropyl)carbamate (348)(450 mg, 1.31 mmol) in DCM (15 mL) was added tri ethyl amine (401 mg,3.936 mmol), 4-nitrophenyl chloroformate (25) (527.405 mg, 2.624 mmol)at 0° C. The resultant reaction mixture was stirred at r.t for 4 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by flashchromatography eluting with 50% EtOAc/hexane to get 0.4 g (60% yield) oftert-buty(2-methyl-2-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)disulfanyl)propyl)carbamate(349) as a sticky liquid. LCMS: MH⁺509, retention time 3.87 min.

4-((1-((Tert-butoxycarbonyl)amino)-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(350): To a stirred solution oftert-buty(2-methyl-2-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)disulfanyl)propyl)carbamate(349) (400 mg, 0.787 mmol) in DMSO (1 ml) was added TEA (239.26 mg, 2.36mmol) and Exatecan mesylate (16) (0.462 mg, 0.785 mmol) at rt. Theresultant reaction mixture was stirred at r.t for 3 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (15 ml) and extractedwith EtOAc (2×20 ml). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound. The crude compound was purified by flash chromatographyeluting with 5% MeOH in DCM to get 0.350 g (75% yield) of compound4-((1-((tert-butoxycarbonyl)amino)-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(350) as an pale brown gum. LCMS: MH⁺805, retention time 3.7 min.

4-((1-Amino-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(351):4-((1-((tert-butoxycarbonyl)amino)-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(350) (0.350 g, 0.435 mmol) in DCM (1 mL) was added TFA (0.2 ml) at 0°C. The resultant reaction mixture was stirred at r.t for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get 0.25 g (80% yield) crude compound4-((1-amino-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(351) as an off-white solid. LCMS: MH⁺805, retention time 3.7 min.

4-((1-Azido-33-methyl-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatetratriacontan-33-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(352): 4-((1-amino-2-methylpropan-2-yl)disulfanyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(351) (0.250 g, 0.492 mmol) HATU (374 mg, 0.984 mmol), azido-peg9-acid(86) (277 mg, 0.541 mmol) and DIPEA (0.181 ml, 0.984 mmol) were mixed inDMF (1 ml) and stirred at ambient temperature under nitrogen atmospherefor 6 h. The progress of the reaction was monitored by LCMS. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound wastriturated with diethyl ether and finally purified by RP-prep-HPLC toobtain the desired product (352) (10.52 mg). ¹H NMR (400 MHz, DMSO-d₆) δ8.12-8.09 (d, 1H) 7.905-7.765 (t, 1H), 7.79-7.77 (d, 1H), 7.559-7.77 (d,2H), 7.43-7.41 (d, 2H), 7.31 (s, 1H), 6.521 (s, 1H), 5.454 (s, 2H), 5.27(s, 3H), 5.12-5.08 (m, 2H), 3.6-3.1 (m, 41H), 2.37 (s, 3H), 2.34-2.131(m, 4H), 1.89-1.87 (m, 2H), 1.15 (s, 6H), 0.85 (t, 3H).LCMS: MH⁺1198,retention time 2.87 min.

Example 59: Synthesis of4-((1-azido-33-methyl-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatetratriacontan-33-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(355)

4-((1-((tert-butoxycarbonyl)amino)-2-methylpropan-2-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(353): To a stirred solution oftert-buty(2-methyl-2-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)disulfanyl)propyl)carbamate(349) (400 mg, 0.787 mmol) in DMSO (1 ml) was added TEA (239.26 mg, 2.36mmol) and compound (105) (460 mg 1.0236 mmol) at r.t. The resultantreaction mixture was stirred at r.t for 3 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was quenched with water (15 ml) and extracted withEtOAc (2×20 ml). The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to get crude compound.The crude compound was purified by flash chromatography eluting with 5%MeOH in DCM to get 0.220 g (85% yield) of compound (353) as an palebrown gum. LCMS: MH⁺819, retention time 3.62 min

4-((1-Amino-2-methylpropan-2-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(354): To a stirred solution of compound4-((1-((tert-butoxycarbonyl)amino)-2-methylpropan-2-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(353) (220 mg, 0.05 mmol) in DCM (1 mL) was added TFA (0.2 ml) at 0° C.The resultant reaction mixture was stirred at r.t for 6 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget 0.15 g (70% yield) of4-((1-amino-2-methylpropan-2-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(354) which was used for next step. LCMS: MH⁺719, retention time 1.33min.

4-((1-azido-33-methyl-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatetratriacontan-33-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(355): 4-((1-amino-2-methylpropan-2-yl)disulfanyl)benzyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(354) (0.150 g, mmol) HATU (158.774 mg, 0.418 mmol), azido-peg9-acid(86) (138.781 mg, 0.272 mmol) and DIPEA (0.074 ml, 0.418 mmol) weremixed in DMF (1 ml) and stirred at ambient temperature under nitrogenatmosphere for 6 h. The progress of the reaction was monitored by LCMS.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound wastriturated with diethyl ether and finally purified by RP-prep-HPLC toobtain the desired product (355) as an off-white solid (15 mg, 7%yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (d, 1H), 7.91 (t, 1H), 7.53 (m,4H), 7.4 (m, 1H), 7.32 (d, 2H), 7.27 (s, 1H), 6.49 (s, 1H), 5.4-5.3 (s,4H), 4.9 (s, 2H), 4.24 (t, 2H), 3.6-3.1 (m, 39H), 2.3 (t, 2H), 1.96 (t,2H), 1.86 (t, 2H), 1.32 (t, 3H), 1.30 (s, 6H), 0.85 (t, 3H). LCMS:MH⁺1213, retention time 2.79 min.

Example 60: Synthesis of1-azido-N-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(366)

35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate (357): To a stirred solution of3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1,35-diol (356)(2.5 g, 4.57 mmol) in DCM (80 mL) was added p-TsCl (0.92 g, 4.80 mmol),silver oxide (1.28 g, 5.48 mmol) and potassium iodide (0.08 g, 0.46mmol) and the reaction mixture was stirred at r.t for 16 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was filtered through celite bed and thefiltrate was concentrated under reduced pressure to get crude compound.The crude compound was purified by column chromatography using silicagel (100-200 mesh) column chromatography eluting with 3% MeOH in DCM toafford 1.8 g (56% yield) of35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate (357) as a yellow liquid. LC-MS: m z 701.55[(M+H)⁺]; R_(t): 1.61 min; 68.17% purity.

35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(358): To a stirred solution of35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate (357) (0.8 g, 1.14 mmol) inN,N-dimethylformamide (8 mL), sodium azide (0.15 g, 2.28 mmol) was addedat rt and the reaction mixture was stirred at 110° C. for 3 h. Reactionwas conducted in two batches (0.8 g scale) of35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate (357) and workup, purification was donetogether. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was cooled to rt andconcentrated under reduced pressure to get crude compound. The crudecompound was dissolved in water (50 mL) and extracted with ethyl acetate(2×50 mL). The combined organic layer was washed with brine solution (20mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to get crude compound. The crude compound was purifiedby silica gel (100-200 mesh) column chromatography eluting with 3% MeOHin DCM to afford 1.3 g (99% yield) of35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(358) as a yellow liquid. ¹H NMR (400 MHz, CDCl₃): Q 4.56 (t, J=5.2 Hz,1H), 3.61-3.53 (m, 44H), 3.48-3.32 (m, 4H).

tert-butyl1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(359): To a stirred solution of35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(358) (1.3 g, 2.27 mmol) in THF (13 mL) potassium tert-butoxide (0.77 g,6.82 mmol) was added at rt and the reaction mixture was stirred at r.tfor 1 h. tert-butyl acrylate (0.58 g, 4.54 mmol) was added and thereaction mixture was stirred at r.t for 2 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was diluted with water (30 mL) and extracted with EtOAc(2×50 mL). The combined organic layer was washed with brine solution (20mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to get crude compound. The crude compound was purifiedby silica gel (100-200 mesh) column chromatography eluting with 4-8%MeOH in DCM to afford 0.9 g (56% yield) of tert-butyl1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(359) as a pale yellow liquid. ¹H NMR (400 MHz, CDCl₃): δ 3.72-3.61 (m,48H), 3.38 (t, J=5.2 Hz, 2H), 2.49 (t, J=6.4 Hz, 2H), 1.44 (s, 9H).

1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid (360): To a stirred solution of tert-butyl1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(359) (0.45 g, 0.64 mmol) in DCM (5 mL), trifluoroacetic acid (3.5 mL)was added at 0° C. and the reaction mixture was stirred at r.t for 1 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was evaporated under reduce pressureto get a crude residue. The residue was diluted with water (10 mL) andextracted with DCM (2×50 mL). The combined organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget 0.3 g (73% yield) of1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid (360) as a yellow gum. ¹H NMR (400 MHz, DMSO-d₆): δ 12.14 (s, 1H),3.61-3.49 (m, 48H), 3.40-3.38 (m, 2H), 2.44 (t, J=6.4 Hz, 2H).

2-methyl-2-(pyridin-2-yldisulfaneyl)propan-1-amine (361): Mixture of1-amino-2-methylpropane-2-thiol hydrochloride (330) (1.0 g, 7.06 mmol)and 1,2-di(pyridin-2-yl)disulfane (328) (3.11 g, 14.12 mmol) in methanol(5.0 mL) was stirred at room temperature for 4 h. The progress of thereaction was monitored by TLC. After completion of starting material,triethylamine (0.71 g, 7.06 mmol) was added to the reaction mixture andthe reaction mixture was stirred at r.t for 5 min. The reaction mixturewas evaporated under reduced pressure to get crude compound. Theobtained crude compound was purified by Davisil grade silica gel columnchromatography using 0 to 70% EtOAc in hexane followed by 0 to 10% MeOHin DCM to afford 1.4 g (93% yield) of2-methyl-2-(pyridin-2-yldisulfaneyl)propan-1-amine (361) as a paleyellow solid. LC-MS: m z 215.26 [(M+H)⁺]; R_(t): 1.17 min; 99.22%purity.

Methyl 2-((1-amino-2-methylpropan-2-yl)disulfaneyl)acetate (363):Mixture of 2-methyl-2-(pyridin-2-yldisulfaneyl)propan-1-amine (361) (1.4g, 6.53 mmol) and methyl 2-mercaptoacetate (362) (1.38 g, 13.06 mmol) inmethanol (20 mL) was stirred at r.t for 16 h and then 90° C. for 4 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was evaporated under reducedpressure to get crude compound. The obtained crude compound was purifiedby Davisil grade silica gel column chromatography using 0 to 70% EtOAcin hexane and then 0 to 10% MeOH in DCM to afford 0.6 g (crude) ofmethyl 2-((1-amino-2-methylpropan-2-yl)disulfaneyl)acetate (363) as ayellow gum. The crude compound was used in the next step without anyfurther purification.

Methyl46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoate(364): Mixture of methyl2-((1-amino-2-methylpropan-2-yl)disulfaneyl)acetate (11) (0.15 g, 0.72mmol),1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid (360) (0.23 g, 0.36 mmol) and HATU (0.33 g, 0.86 mmol) inN,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (0.28 g, 2.16mmol) was added at 0° C. and the reaction mixture was stirred at r.t for4 h. The progress of the reaction was monitored by TLC. After completionof starting material, the reaction mixture was diluted with ice coldwater (20 mL) and extracted with ethyl acetate (3×30 mL). The combinedorganic extract was dried over anhydrous Na₂SO₄, filtered, evaporatedunder reduced pressure to get crude compound. The obtained crudecompound was purified by silica gel (100-200 mesh) column chromatographyeluting with 5% MeOH in DCM to afford 0.24 g (crude) of methyl46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoate(364) as a yellow gum. The crude compound was used in the next stepwithout any further purification.

46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoicacid (365): To a stirred solution of methyl46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoate(364) (0.17 g, 0.20 mmol) in THF (5 mL), LiOH·H₂O (0.043 g, 1.02 mmol)in water (5 mL) was added and the reaction mixture was stirred at r.tfor 4 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was diluted withice cold water (20 mL) and extracted with diethyl ether (3×30 mL). Theseparated aqueous layer was acidified with glacial acetic acid at 0° C.and resulting acidified layer was extracted with ethyl acetate (3×30mL). The combined organic extract was dried over anhydrous Na₂SO₄,filtered and evaporated under reduced pressure to get 0.09 g (54% yield)of46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoicacid (365) as a yellow gum. LC-MS (ELSD): m/z 821.3 [(M+H)⁺]; R_(t):3.092 min; 69.37% purity.

1-azido-N-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(366): To a stirred suspension of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (0.037 g, 0.07 mmol),46-azido-5,5-dimethyl-8-oxo-11,14,17,20,23,26,29,32,35,38,41,44-dodecaoxa-3,4-dithia-7-azahexatetracontanoicacid (365) (0.09 g, 0.11 mmol) and PyBOP (0.086 g, 0.16 mmol) inN,N-dimethylformamide (5 mL), DIPEA (0.043 g, 0.33 mmol) was added at rtand the reaction mixture was stirred at r.t for 16 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was diluted with ice cold water (20 mL)and extracted with ethyl acetate (3×30 mL). The combined organic extractwas dried over anhydrous Na₂SO₄, filtered and evaporated under reducedpressure to get crude compound. The obtained crude compound was purifiedby reverse phase preparative HPLC to afford 0.0049 g (7% yield) of1-azido-N-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(366) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.68 (t, J=8.5Hz, 1H), 7.88 (t, J=6.4 Hz, 1H), 7.82 (d, J=9.1 Hz, 1H), 7.31 (s, 1H),6.52 (s, 1H), 5.57-5.56 (m, 1H), 5.43 (s, 2H), 5.29 (q, J=20.1 Hz, 2H),3.61-3.48 (m, 50H), 3.18 (d, J=6.1 Hz, 4H), 2.50 (s, 2H), 2.42 (s, 3H),2.33 (t, J=6.2 Hz, 2H), 2.24-2.11 (m, 2H), 1.90-1.82 (m, 2H), 1.13 (s,6H), 0.87 (t, J=7.2 Hz, 3H). LC-MS (method 25): m z 1238.69 [(M+H)⁺];R_(t): 1.92 min; 96.15% purity, HP-LC (method 25): R_(t): 5.60 min;96.84% purity.

Example 61: Synthesis of37-Azido-8-oxo-11,14,17,20,23,26,29,32,35-nonaoxa-3,4-dithia-7-azaheptatriacontyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(370)

2-(Pyridin-2-yldisulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(367): To a stirred solution 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl) carbonate (337) (282.21 mg, 0.801mmol) in DMSO (5 mL) was added TEA (0.18 ML, 1.335 mmol), and(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105) (300 mg, 0.667 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16h. The progress of the reaction was monitored byLCMS. After completion of starting material, reaction mixture wasquenched with water (15 mL) and extracted with 10% methanol in DCM (2×10mL). The combined organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. To the crude material diethyletherwas added and solid precipitated out. It was filtered and the solidmaterial was passed through Combi-flash column chromatography elutingwith 5% MeOH in DCM to get 0.330 g (74% yield) of2-(pyridin-2-yldisulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(367) as an off-white solid. LCMS: MH⁺663, retention time 3.25 min.

2-((2-((Tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(368): To a stirred solution of tert-butyl (2-mercaptoethyl)carbamate(339) (132.38 mg, 0.747 mmol) in MeOH (5 ml) was added2-(pyridin-2-yldisulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(367) (330 mg, 0.498 mmol) and the reaction mixture was degassed withargon for 15 min. The resultant reaction mixture was stirred at r.t for2 h. The progress of the reaction was monitored by TLC. After completionof starting material, the reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby flash chromatography eluting with 5% MeOH in DCM to get 0.270 g (74%yield) of 2-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(368) as a light yellow gum. LCMS: MH⁺729, retention time 3.27 min.

2-((2-Aminoethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(369): To a stirred solution of2-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(368) (270 mg, 0.37 mmol) in DCM 5 ml TFA (1 ml) was added at 0° C. andthe resultant reaction mixture was stirred at r.t for 1 h. The progressof the reaction was monitored by LCMS. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was triturated with diethyl etherto obtain 0.230 g (98% yield) of 2-((2-aminoethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(369) as an off-white solid. LCMS: MH⁺628, retention time 1.53 min.

37-azido-8-oxo-11,14,17,20,23,26,29,32,35-nonaoxa-3,4-dithia-7-azaheptatriacontyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(370): To a stirred solution of 2-((2-aminoethyl)disulfanyl)ethyl(S)-(3-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)carbamate(369) (200 mg, 0.318 mmol), in DMF (5 mL) was added DIPEA (0.139 mL,0.795 mmol), HATU (241.88 mg, 0.636 mmol) and azido-(PEG)₉-acid (86)(195.26 mg, 0.382 mmol) at 0° C. The resultant reaction mixture wasstirred at r.t for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. It waspurified by normal column chromatography followed by RP-prep-HPLC toobtain the desired product (370) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆): δ 8.02-8.08 (m, 2H), 7.48-7.51 (d, 2H), 7.36 (s, 1H), 7.26 (s,1H), 6.50 (s, 1H), 5.42 (s, 2H), 5.30 (s, 2H), 4.16-4.24 (m, 4H),3.46-3.59 (m, 37H), 3.18-3.39 (m, 6H), 2.91-2.94 (t, 2H), 2.74-2.78 (t,2H), 2.28-2.31 (t, 2H), 1.82-1.97 (m, 4H), 1.17-1.33 (m, 6H), 0.85-0.88(t, 3H). LCMS: MH⁺1122, retention time 1.65 min.

Example 62: Synthesis of1-azido-N-(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(378)

2-(pyridin-2-yldisulfaneyl)ethan-1-amine (372): A mixture of2-aminoethane-1-thiol (371) (0.2 g, 2.59 mmol) and1,2-di(pyridin-2-yl)disulfane (328) (0.68 g, 3.11 mmol) in MeOH (2.0 mL)was stirred at r.t for 16 h. The progress of the reaction was monitoredby TLC. After completion of starting material, the reaction mixture wasevaporated under reduced pressure to get crude compound. The obtainedcrude compound was purified by Davisil grade silica gel columnchromatography using 16 to 18% MeOH in DCM to afford 0.2 g (41% yield)of 2-(pyridin-2-yldisulfaneyl)ethan-1-amine (372) as a yellow gum.LC-MS: m z 187.16 [(M+H)⁺]; R_(t): 0.89 min; 61.49% purity.

Tert-butyl (2-((2-aminoethyl)disulfaneyl)-2-methylpropyl)carbamate(373): A mixture of 2-(pyridin-2-yldisulfaneyl)ethan-1-amine (372) (0.35g, 1.88 mmol) and tert-butyl (2-mercapto-2-methylpropyl)carbamate (347)(0.38 g, 1.88 mmol) in MeOH (10.0 mL) was stirred at r.t for 4 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was evaporated under reducedpressure to get crude compound. The obtained crude compound was purifiedby Davisil grade silica gel column chromatography using 15% MeOH in DCMto afford 0.4 g (76% yield) of tert-butyl(2-((2-aminoethyl)disulfaneyl)-2-methylpropyl)carbamate (373) as ayellow solid. ¹H NMR (400 MHz, DMSO-d₆): 8 ppm: 6.98 (s, 1H), 3.09 (d,J=6.4 Hz, 2H), 2.80-2.71 (m, 4H), 1.89 (s, 2H), 1.39 (s, 9H), 1.78 (s,6H).

Tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(375): To a stirred solution of tert-butyl(2-((2-aminoethyl)disulfaneyl)-2-methylpropyl)carbamate (373) (0.41 g,1.46 mmol) in THF (15.0 mL), DIPEA (2.55 mL, 14.62 mmol) was addedfollowed by the addition of 2,4,6-trichloro-1,3,5-triazine (374) (0.32g, 1.75 mmol) at 0° C. and the reaction mixture was stirred at 0° C. for1 h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was quenched with water (20 mL)and extracted with ethyl acetate (3×30 mL). The combined organic layerwas dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude compound. The obtained crude compound was purifiedby silica gel column chromatography using 15% EtOAc in hexane to afford0.38 g (61% yield) of tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(375) as pale yellow gum. LC-MS: m z 426.03 [(M−H)⁻]; R_(t): 2.27 min;99.74% purity.

Tert-butyl(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(376): To a stirred suspension of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (0.05 g, 0.094 mmol) in THF/DCM (10.0 mL, 1:1 byvolume) tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(375) (0.045 g, 0.105 mmol) and DIPEA (0.18 mL, 1.05 mmol) was added andthe reaction mixture was stirred at r.t for 2 h. tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(375) (0.023 g, 0.052 mmol) was again added and the reaction mixture washeated at 80° C. for 48 h. After completion of starting material,reaction mixture was cooled to rt, diluted with water (20 mL) andextracted with ethyl acetate (2×30 mL). The combined organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude compound. The obtained crude compound was purifiedby silica gel (100-200 mesh) column chromatography using 4% MeOH in DCMto afford 0.06 g (77% yield) of tert-butyl(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(376) as a yellow solid. LC-MS: m z 827.08 [(M+H)⁺]; R_(t): 2.29 min;92.25% purity.

(1S,9S)-1-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione2,2,2-trifluoroacetate (377): To a stirred solution of tert-butyl(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(376) (0.06 g, 0.072 mmol) in DCM (5.0 mL), TFA (2.0 mL) was added at 0°C. and the reaction mixture was stirred at 0° C. for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was evaporated under reduced pressure toget a crude residue. The resulting residue was washed with diethyl ether(3×10 mL), n-pentane (10 mL) and dried under vacuum to afford 0.06 g(crude) compound. 0.015 g of crude material was purified by RPpreparative HPLC to afford 0.0095 g (16% yield) of(1S,9S)-1-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione2,2,2-trifluoroacetate (377) as a yellow solid. ¹H NMR (400 MHz,DMSO-d₆): δ 8.67-8.47 (m, 1H), 8.16-8.13 (m, 1H), 7.81-7.74 (m, 4H),7.31 (s, 1H), 6.51 (d, J=4.2 Hz, 1H), 5.73 (s, 1H), 5.40 (s, 2H),5.23-5.13 (m, 2H), 3.55-3.45 (m, 2H), 3.17-2.51 (m, 6H), 2.40 (s, 3H),2.29 (s, 2H), 1.88-1.83 (m, 2H), 1.36-1.30 (m, 3H), 1.14-1.10 (m, 3H),0.87 (t, J=7.3 Hz, 3H). LC-MS (method 31): m/z 727.06 [(M+H)⁺]; R_(t):2.17 min; 98.11% purity, HP-LC (method 31): VIIR_(t): 3.69 min; 98.20%purity.

1-azido-N-(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(378): To the stirred solution of(1S,9S)-1-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione2,2,2-trifluoroacetate (377) (35 mg, 0.04 mmol) in DMF (5 mL) was added1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid (360) (32 mg, 0.05 mmol), PyBOP (31 mg, 0.06 mmol) and DIPEA (0.03mL, 0.17 mmol) at 0° C. and the reaction mixture was stirred at r.t for3 h. The progress of the reaction was monitored by TLC. After completionof starting material, the reaction mixture was diluted with ice coldwater (20 mL) and extracted with ethyl acetate (30 mL×3). The combinedorganic extract was dried over anhydrous Na₂SO₄ and evaporated underreduced pressure to get crude compound. The obtained crude compound waspurified by davisil silica gel column chromatography using 5% methanolin dichloromethane and further purified by reverse phase preparativeHPLC to afford 6.4 mg (11% yield) of1-azido-N-(2-((2-((4-chloro-6-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(378) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.64-8.44 (m,1H), 8.12-8.03 (m, 1H), 7.87-7.75 (m, 2H), 7.31 (s, 1H), 6.50 (d, J=9.2Hz, 1H), 5.75-5.61 (m, 1H), 5.40 (s, 2H), 5.25-5.06 (m, 2H), 3.51-3.12(m, 52H), 3.20-2.67 (m, 8H), 2.39 (s, 3H), 2.33-2.25 (m, 2H), 1.88-1.84(m, 2H), 1.21-1.15 (m, 3H), 0.98 (s, 3H), 0.89 (t, J=7.2 Hz, 3H). LC-MS(method 36): m/z 1352.70 [(M+H)⁺]; R_(t): 2.01 min; 95.26% purity, HP-LC(method 36): R_(t): 4.74 min; 96.29% purity.

Example 63: Synthesis of(S)-9-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(381) (TFA salt)

tert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(379): To the stirred solution of tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(375) (150 mg, 0.35 mmol) in DMF (10 mL) was added DIPEA (0.12 mL, 0.70mmol) and(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (137 mg, 0.35 mmol) slowly at 0° C. and the reaction mixture washeated at 60° C. for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure and quenched with ice cold water (30mL) and extracted with EtOAc (2×40 mL). The combined organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure toget 100 mg (crude) of tert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(379) as a pale brown gum. The crude compound was used in the next stepwithout any further purification. LC-MS: m z 784.06 [(M+H)⁺]; R_(t):2.16 min; 83.32% purity.

(S)-9-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(380): To a stirred solution of tert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)-2-methylpropyl)carbamate(379) (35 mg, 0.04 mmol) in dichloromethane (5 mL) 2,2,2-trifluoroaceticacid (1 mL) was added at 0° C. and the reaction mixture was stirred atroom temperature for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, the reaction mixture wasevaporated under reduced pressure to get crude compound. The crudecompound was purified by RP-Prep HPLC to afford 10 mg (33% yield) of(S)-9-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(380) (TFA salt) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.91-8.72 (m, 1H), 8.25-8.22 (m, 1H), 8.17-8.15 (m, 1H), 7.84-7.77 (m,3H), 7.34 (s, 1H), 6.54 (d, J=2.4 Hz, 1H), 5.45 (s, 2H), 5.35 (d, J=2.8Hz, 2H), 3.57-3.39 (m, 2H), 3.20 (t, J=6.4 Hz, 2H), 2.97-2.77 (m, 4H),1.90-1.86 (m, 2H), 1.33-1.27 (m, 6H), 1.18 (s, 3H), 0.89 (t, J=7.2 Hz,3H). LC-MS (method 36): m/z 682.27 [(M−H)⁻]; R_(t): 1.53 min; 95.27%purity, HP-LC R_(t): 3.20 min; Purity: 96.46%.

(S)-1-azido-N-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(381): To a stirred solution of(S)-9-((4-((2-((2-aminoethyl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(380) (100 mg, 0.15 mmol),1-azido-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid (360) (115 mg, 0.18 mmol) and T₃P (0.19 mL, 0.063 mmol) inN,N-dimethylformamide (2 mL), TEA (0.06 mL, 0.45 mmol) was added at 0°C. and the reaction mixture was stirred at room temperature for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was diluted with ice cold water(20 mL) and extracted with ethyl acetate (3×30 mL). The combined organicextract was dried over anhydrous Na₂SO₄ and evaporated under reducedpressure to get crude compound. The obtained crude compound was purifiedby preparative HPLC to afford 27.4 mg (14% yield) of(S)-1-azido-N-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide(381) as a colorless semi-solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.87-8.69(m, 1H), 8.24-8.21 (m, 1H), 8.16 (d, J=1.6 Hz, 1H), 8.02-7.93 (m, 1H),7.82-7.78 (m, 1H), 7.34 (s, 1H), 6.52 (s, 1H), 5.45 (s, 2H), 5.35 (s,2H), 3.60-3.38 (m, 52H), 3.19 (d, J=6.4 Hz, 1H), 2.86-2.71 (m, 4H), 2.54(s, 2H), 2.31-2.22 (m, 2H), 1.89-1.85 (m, 2H), 1.28 (q, J=7.6 Hz, 4H),0.88 (t, J=7.2 Hz, 3H). LC-MS (method 28): m/z 1281.76 [(M+H)⁺]; R_(t):1.88 min; 96.35% purity: R_(t): 4.04 min; 96.37% purity.

Example 64: Synthesis of(S,Z)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(392)

tert-butyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate (382): To astirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (1.5 g, 3.82 mmol), in DMF (10 mL) and acetone (20 mL) was addedtert-butyl 2-bromoacetate (745 mg, 3.82 mmol) followed by potassiumcarbonate (792 mg, 5.73 mmol) under nitrogen atm at rt. The resultantreaction mixture was stirred at 80° C. for 3 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was cooled to 0° C., quenched with ice cold water (30mL). The reaction mixture was stirred for 15 min, filtered, trituratedwith acetone to get 1.3 g (67% yield) oftert-butyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(382) as a pale yellow solid. The crude compound was used in the nextstep without any further purification. LC-MS: m z 507.81 [(M+H)⁺];R_(t): 1.91 min; 87.40% purity.

(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)aceticacid (383): To a stirred solution oftert-butyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate(382) (1.3 g, 2.57 mmol), in DCM (13 mL) was added trifluoroacetic acid(13 mL) at 0° C. The resultant reaction mixture was stirred at rt for 3h. The progress of the reaction was monitored by LCMS and TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. This crude compound wastriturated with diethyl ether to get 1.1 g (95% yield) of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)aceticacid (383) as a yellow solid. The crude compound was used in the nextstep without any further purification. LC-MS: m z 451.31 [(M+H)⁺];R_(t): 1.35 min; 97.92% purity.

tert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetyl)hydrazine-1-carboxylate(384): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1Hpyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)aceticacid (383) (1.1 g, 2.44 mmol) in DMF (6 mL) was added triethylamine (1.0mL, 7.32 mmol) and T3P (6.2 mL, 9.76 mmol) at 0° C. and the reactionmixture was stirred at 0° C. for 30 min. NH₂NHBoc (806 mg, 6.10 mmol)was added to the reaction mixture at 0° C. under nitrogen atmosphere Theresultant reaction mixture was stirred at rt for 16 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was quenched with water (20 mL) and extractedwith EtOAc (2×200 mL). The combined organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudecompound. This crude compound was triturated with diethyl ether to get900 mg (65% yield) of tert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetyl)hydrazine-1-carboxylate(384) as a pale yellow solid. The crude compound was used in the nextstep without any further purification. LC-MS: m z 565.89 [(M+H)⁺];R_(t): 1.57 min; 91.36% purity.

(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-yrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385): A stirred solution oftert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetyl)hydrazine-1-carboxylate(384) (300 mg, 0.53 mmol) in DCM (6 mL) was cooled to 0° C. 4M HCl in 1,4-dioxane (3 mL) was added to the reaction mixture at 0° C. undernitrogen atmosphere The reaction mixture was stirred at rt for 3 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was washed withdiethyl ether (20 mL), dissolved in methanol (25 mL), added carbonate onpolymer support resin (150 mg) and the reaction mixture was stirred atrt for 30 min. The reaction mixture was filtered, concentrated underreduced pressure to get 320 mg of crude(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-yrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385). 100 mg crude (5) was taken for purification by RP-prep HPLC toget 7 mg of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-yrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.52 (s, 1H),8.11 (d, J=9.2 Hz, 1H), 7.61-7.54 (m, 2H), 7.28 (s, 1H), 6.50 (s, 1H),5.43 (s, 2H), 5.32 (s, 2H), 4.75 (s, 2H), 4.38 (s, 2H), 3.19 (q, J=7.4Hz, 2H), 1.90-1.83 (m, 2H), 1.31 (t, J=7.5 Hz, 3H), 0.88 (t, J=7.3 Hz,3H). LC-MS (method 10): m/z 465.30 [(M+H)⁺]; R_(t): 1.63 min; 96.02%purity; HP-LC (method 10): R_(t): 4.01 min; 97.13% purity.

17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate(387): To a stirred solution of3,6,9,12,15-pentaoxaheptadecane-1,17-diol (386) (2.5 g, 8.85 mmol), inDCM (10 mL) was added silver oxide (3.13 g, 13.5 mmol) followed bypotassium iodide (294 mg, 1.77 mmol) under nitrogen atm at rt. Theresultant reaction mixture was stirred at rt for 10 min. p-TsCl (1.79 g,9.41 mmol) was added and the resultant reaction mixture was stirred atrt for 30 min. The progress of the reaction was monitored by TLC andLCMS. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (230-400mesh) eluting with 3% MeOH-DCM to get 2 g (52% yield) of17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (387)as a colorless gum. ¹H NMR (400 MHz, DMSO-d₆): δ 7.80 (d, J=8.4 Hz, 2H),7.34 (d, J=8.0 Hz, 2H), 4.18-3.58 (m, 25H), 3.58 (s, 3H).

17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (388): To a stirredsolution of 17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl4-methylbenzenesulfonate (387) (2 g, 4.58 mmol), in DMF (20 ml) wasadded sodium azide (447 mg, 6.87 mmol) at rt. The resultant reactionmixture was heated at 100° C. for 3 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was concentrated under reduced pressure to get crude compound.The crude compound was dissolved in water (50 mL) and extracted withEtOAc (2×200 mL). The combined organic layer was washed with brinesolution (20 mL) and dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to get crude compound. The crude compound wastriturated with diethyl ether to get 1 g (71% yield) of17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (388) as a colorless gum.The crude compound was used in the next step without any furtherpurification. ¹H NMR (400 MHz, DMSO-d₆): δ 3.73-3.06 (m, 24H), 3.05 (s,1H).

17-azido-3,6,9,12,15-pentaoxaheptadecylmethanesulfonate (389): To astirred solution of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (388)(1 g, 3.25 mmol) in DCM (6 mL) was added triethylamine (1.36 mL, 9.75mmol) followed by methanesulphonyl chloride (1.26 mL, 16.27 mmol) at 0°C. under nitrogen atmosphere The resultant reaction mixture was stirredat rt for 2 h. The progress of the reaction was monitored by TLC andLCMS. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was dissolved in water (50 mL) and extracted with EtOAc (2×200mL). The combined organic layer was washed with saturated NaHCO₃solution (30 mL), brine (30 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get to get 1.14 g (91% yield) of17-azido-3,6,9,12,15-pentaoxaheptadecylmethanesulfonate (389) as acolorless gum. ¹H NMR (400 MHz, DMSO-d₆): δ 4.40-4.37 (m, 2H) 3.78-3.76(m, 2H), 3.69-3.63 (m, 18H) 3.39 (t, J=5.2 Hz, 2H) 3.08 (s, 3H).

1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(391): To a stirred solution of17-azido-3,6,9,12,15-pentaoxaheptadecylmethanesulfonate (389) (0.50 g,1.30 mmol) and 1-(4-hydroxyphenyl)ethan-1-one (390) (177 mg, 1.30 mmol)in ACN (6 mL) was added potassium carbonate (0.268 g, 1.95 mmol) to thereaction mixture at 0° C. under nitrogen atmosphere The resultantreaction mixture was stirred at 70° C. for 16 h. The progress of thereaction was monitored by TLC and LCMS. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was dissolved in water (50 mL)and extracted with EtOAc (2×100 mL). The combined organic layer waswashed with brine (50 mL), dried over anhydrous Na₂SO₄, concentratedunder reduced pressure to get crude compound. The crude compound waspurified by column chromatography using silica gel (60-120 mesh) elutingwith 3% MeOH-DCM to get 220 mg (40% yield) of1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl) oxy) phenyl)ethan-1-one(391) as a colorless gum. LC-MS: m z 426.32 [(M+H)⁺]; R_(t): 1.69 min;94.21% purity.

(S,E)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide&(S,Z)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(392): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385) (200 mg, 0.43 mmol) in ethanol (8 mL) was added1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(391) (200 mg, 0.47 mmol) and the reaction mixture was stirred at 80° C.for 6 h under Nitrogen atmosphere The progress of the reaction wasmonitored by TLC and LCMS. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude compound was purified by SFC purification. Thepurified fractions were concentrated by lyophilisation to get 27 mg (7%yield) of(S,E)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide&(S,Z)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(392) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.79 (s, 1H),8.11 (d, J=8.8 Hz, 1H), 7.83-7.75 (m, 2H), 7.64 (s, 0.5H), 7.53-7.47 (m,2H), 7.28 (s, 0.4H), 6.98 (d, J=8.8 Hz, 2H), 6.50 (s, 0.3H), 6.20 (s,0.55H), 5.71 (s, 0.57H), 5.44 (d, J=8.7 Hz, 2H), 5.29 (s, 0.8H), 5.18(s, 1.2H), 5.00 (d, J=9.4 Hz, 0.6H), 4.90 (q, J=6.5 Hz, 0.6H), 4.63 (t,J=5.7 Hz, 0.6H), 4.13 (t, J=4.2 Hz, 2H), 3.75 (t, J=4.2 Hz, 2H),3.57-3.50 (m, 18H), 3.17-3.06 (m, 4H), 2.32-2.27 (m, 3H), 2.13-2.01 (m,0.7H), 2.01-1.88 (m, 1H), 1.87-1.84 (m, 1H), 1.27-1.23 (m, 4H),0.89-0.83 (m, 3H). LC-MS (method 13): m/z 872.1 [(M+H)⁺]; R_(t): 2.33,2.63 min; 57.72%+37.34% purity, HP-LC (method 13): R_(t): 5.64, 6.25min; Purity: 58.53%+36.75%.

Example 65: Synthesis of(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(398)

35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate (394): To a stirred solution of3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1,35-diol (393)(2.5 g, 4.57 mmol) in DCM (80 mL) was added silver oxide (1.27 g, 5.48mmol) followed by potassium iodide (76 mg, 0.46 mmol) at rt undernitrogen atmosphere The resultant reaction mixture was stirred at rt for10 min. p-TsCl (915 mg, 4.80 mmol) was added and the resultant reactionmixture was stirred at rt for 24 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, the reactionmixture was filtered through celite bed and the filtrate wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (230-400mesh) eluting with 3% methanol in dichloromethane to get 1.6 g (50%yield) of35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzene sulfonate (394) as a colorless gum. LC-MS: m z 701.12[(M+H)⁺]; R_(t): 1.61 min; 76.60% purity.

35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(395): To a stirred solution of35-hydroxy-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl4-methylbenzenesulfonate(394) (1.6 g, 2.28 mmol) in DMF (10 mL) was added sodium azide (0.3 g,4.56 mmol) at rt. The resultant reaction mixture was stirred at 100° C.for 16 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was dissolvedin water (50 mL), extracted with EtOAc (2×50 mL) and washed with brinesolution (20 mL). The combined organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to get 0.85 g (65% yield)of 35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(395) as a colorless gum. ¹H NMR (400 MHz, CDCl₃): δ 3.72-3.59 (m, 46H),3.39 (t, J=5.2 Hz, 2H), 2.91 (s, 1H).

35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontylmethanesulfonate (396): To a stirred solution of35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-ol(395) (0.30 g, 0.52 mmol) in DCM (6 mL) was added triethylamine (0.22mL, 1.57 mmol) followed by methanesulphonyl chloride (0.10 mL, 1.30mmol) at 0° C. under nitrogen atmosphere The resultant reaction mixturewas stirred at 0° C. to rt for 2 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was diluted with DCM (20 mL). The organic layer was washed withsaturated NaHCO₃ solution (2×20 mL) followed by brine (20 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to get 0.3 g (crude) of35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl methanesulfonate (396) as a colorless gum. The crude compound was used in thenext step without any further purification.

1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethan-1-one(397): To a stirred solution of35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontylmethanesulfonate (396) (0.3 g, 0.46 mmol) in acetonitrile (6 mL) wasadded potassium carbonate (0.94 g, 0.69 mmol) followed by1-(4-hydroxyphenyl)ethan-1-one (390) (95 mg, 0.70 mmol) at rt undernitrogen atmosphere The resultant reaction mixture was stirred at 80° C.for 6 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was dissolvedin water (30 mL) and extracted with EtOAc (2×50 mL), washed with brinesolution (50 mL). The combined organic layer was dried over anhydrousNa₂SO₄, concentrated under reduced pressure to get crude compound. Thecrude compound was purified by column chromatography using silica gel(60-120 mesh) eluting with 3-5% methanol in dichloromethane to get 0.25g (79% yield) of1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethan-1-one(397) as a colorless gum. LC-MS: m z 691.09 [(M+H)⁺]; R_(t): 1.73 min;69.80% purity.

(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(398): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385), (0.2 g, 0.43 mmol) in ethanol (10 mL) was added1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethan-1-one(397) (235 mg, 0.34 mmol). Then the resultant reaction mixture wasstirred at 90° C. for 3 h. The progress of the reaction was monitored byTLC and LCMS. After completion of starting material, reaction mixturewas concentrated under reduced pressure to get crude compound. The crudecompound was purified by SFC purification to afford 14.5 mg (3% yield)of(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(398) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.79 (d,J=83.2 Hz, 1H), 8.12 (d, J=9.1 Hz, 1H), 7.83-7.74 (m, 2H), 7.64 (s, 1H),7.55-7.47 (m, 2H), 6.98 (d, J=8.7 Hz, 2H), 6.21 (s, 1H), 5.72 (d, J=4.6Hz, 1H), 5.43 (s, 1H), 5.19 (s, 2H), 4.98 (s, 0.5H), 4.90 (t, J=9.8 Hz,1H), 4.62 (dd, J=11.6, 4.0 Hz, 1H), 4.13 (d, J=4.2 Hz, 2H), 3.75 (s,2H), 3.60-3.49 (m, 42H), 3.16-3.06 (m, 4H), 2.32-2.27 (m, 3H), 2.14-2.11(m, 1H), 2.02-1.99 (m, 1H), 1.24 (q, J=7.9 Hz, 3H), 0.85 (t, J=7.1 Hz,3H). LC-MS (method 5): m/z 1136.49 [(M+H)⁺]; R_(t): 1.57, 1.84 min;94.70+3.67% purity, HP-LC (method 5): R_(t): 3.51, 4.03 min; 90.53+4.11%purity.

Example 66: Synthesis of(S,E)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(403)

1-(4-hydroxyphenyl)-2-methylpropan-1-one (400): To a stirred solution ofphenol (399) (5 g, 53.13 mmol), in DCM (30 mL) was added isobutyrylchloride (5.9 g, 55.79 mmol) followed by aluminium chloride (17.71 g,132.83 mmol) under nitrogen atm at 10° C. The resultant reaction mixturewas stirred at rt for 16 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wascooled to 0° C., quenched with ice cold water (100 mL) and pH=11 wasadjusted by the addition of 30% aqueous NaOH solution. The aqueous layerwas washed with diethyl ether and separated. Then aqueous layer pH=1 wasadjusted by addition of 20% aqueous H₂SO₄ solution and extracted withdiethylether (3×70 mL). Combined organic layer was washed with brine (50mL) and dried over anhydrous Na₂SO₄, concentrated under reduced pressureto get crude compound. The crude compound was purified by columnchromatography using silica gel (230-400 mesh) eluting with 15% EtOAc inPet ether to get 3.5 g (40% yield) of1-(4-hydroxyphenyl)-2-methylpropan-1-one (400) as a colorless oil. LCMS:m/z 165.70 [(M+H)⁺]; R_(t): 1.50 min; 98.09% purity.

(Z)-4-(1-hydrazineylidene-2-methylpropyl)phenol (401): To a stirredsolution of 1-(4-hydroxyphenyl)-2-methylpropan-1-one (400) (500 mg, 3.05mmol) in ethanol (20 mL) was added hydrazine hydrate (763 mg, 15.25mmol) followed by acetic acid (2 drops) under nitrogen atm at rt. Theresultant reaction mixture was stirred at 80° C. for 16 h. The progressof the reaction was monitored by LCMS and TLC. After completion ofstarting material, the reaction mixture was concentrated under reducedpressure, quenched with ice cold water (20 mL) and extracted with EtOAc(2×50 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to get 500 mg (93% Yield) of(Z)-4-(1-hydrazineylidene-2-methylpropyl)phenol (401) as an off-whitesolid. LC-MS: m z 179.22 [(M+H)⁺)]; R_(t): 1.05 min; 76.70% purity.

(S,E)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N′-(1-(4-hydroxyphenyl)-2-methylpropylidene)acetohydrazide (402): To a stirred solution of(Z)-4-(1-hydrazineylidene-2-methylpropyl)phenol (401) (100 mg, 0.56mmol) in DMF (15 mL) was added triethylamine (0.24 mL, 1.68 mmol) andT₃P (0.72 mL, 1.12 mmol) under nitrogen atm at 0° C.(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino [1,2-b]quinolin-9-yl)oxy)acetic acid (383) (252 mg, 0.56mmol) was added to the reaction mixture under nitrogen atm at 0° C. Theresultant reaction mixture was stirred at 0° C. to rt for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was quenched with 5% saturateNaHCO₃ solution (20 mL) and stirred for 15 min. The precipitated solidwas filtered off, dried under vacuum and triturated with diethyl etherto get 180 mg (52% Yield) of(S,E)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N′-(1-(4-hydroxyphenyl)-2-methylpropylidene)acetohydrazide(402) as a light yellow solid. LC-MS: m z 611.33 [(M+H)⁺)]; R_(t): 1.72min; 82.43% purity.

(S,E)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(403): To a stirred solution of(S,E)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N′-(1-(4-hydroxyphenyl)-2-methylpropylidene)acetohydrazide(402) (150 mg, 0.25 mmol) in acetonitrile (30 mL) was added potassiumcarbonate (52 mg, 0.38 mmol) followed by17-azido-3,6,9,12,15-pentaoxaheptadecyl methanesulfonate (389) (96 mg,0.25 mmol) under nitrogen atm at 0° C. The resultant reaction mixturewas stirred at 70° C. for 36 h. The progress of the reaction wasmonitored by TLC and LCMS. After completion of starting material, thereaction mixture was concentrated under reduced pressure, quenched withice cold water (50 mL) and extracted with EtOAc (2×50 mL). The combinedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to get crude compound. The crude compound was purifiedby SFC purification to afford 14 mg (6% yield) of(S,E)-N′-(1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(403) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.55 (d,J=13.6 Hz, 1H), 8.10 (t, J=11.6 Hz, 1H), 7.55-7.40 (m, 1.55H), 7.28 (s,1H), 7.22-7.14 (m, 2.31H), 7.07 (t, J=11.2 Hz, 1.88H), 6.49 (s, 1H),5.42 (s, 2H), 5.31 (s, 2.74H), 4.81 (s, 1H), 4.15 (t, J=4 Hz, 2H), 3.79(s, 2H), 3.61-3.48 (m, 18H), 3.31 (m, 2H), 3.16-3.14 (m, 2H), 2.85-2.81(m, 1H), 1.90-1.83 (m, 2H), 1.29-1.25 (m, 3H), 1.11-1.04 (m, 6H), 0.89(m, 3H). LC-MS (method 7): m/z 900.45 [(M+H)⁺]; R_(t): 4.55, 4.37 min;82.01+10.80% purity, HP-LC (method 7): R_(t): 4.44, 4.27 min;83.45+10.06% purity. (This is a mixture of E and Z isomers)

Example 67: Synthesis of3-(2-((E)-1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazinyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide&3-(2-((Z)-1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide(411)

3-ethoxy-2,2-dimethyl-3-oxopropanoic acid (405): To a stirred solutionof diethyl 2,2-dimethylmalonate (404) (3.0 g, 15.94 mmol) in ethanol (30mL) was added KOH (0.89 g, 15.94 mmol) at 0° C. and stirred for 16 h atrt. The progress of the reaction was monitored by TLC. After completion,the reaction mixture was concentrated under reduced pressure to get acrude residue. The residue was diluted with water (20 mL) and washedwith ethyl acetate. The aqueous layer was separated, acidified with 1NHCl and extracted with EtOAc (2×50 mL). The combined organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get 1.44 g (56% yield) of(3-ethoxy-2,2-dimethyl-3-oxopropanoic acid (405) as a colorless liquid.¹H NMR (400 MHz, DMSO-d₆): δ 12.75 (s, 1H), 4.10 (q, J=7.2 Hz, 2H), 1.27(s, 9H), 1.16 (t, J=7.2 Hz, 3H).

tert-butyl2-(3-ethoxy-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate (407):To a stirred solution of 3-ethoxy-2,2-dimethyl-3-oxopropanoic acid (405)(0.800 g, 4.99 mmol), tert-butyl hydrazinecarboxylate (406) (0.66 g,4.99 mmol) in DMF (10 mL) was added TEA (2.1 mL, 14.99 mmol) followed byT₃P (4.8 mL, 7.49 mmol, 50% solution in EtOAc) at RT. The resultantreaction mixture was stirred for 2 h at r.t. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was cooled to 0° C., quenched with H₂O (10 mL) andextracted with EtOAc (2×25 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to get0.85 g (62% yield) of tert-butyl2-(3-ethoxy-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate (407) asan off-white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.27 (s, 1H), 6.44 (s,1H), 4.21 (q, J=7.2 Hz, 2H), 1.50 (s, 6H), 1.47 (s, 9H), 1.29 (t, J=7.2Hz, 3H).

3-(2-(tert-butoxycarbonyl)hydrazineyl)-2,2-dimethyl-3-oxopropanoic acid(408): To a stirred solution of tert-butyl2-(3-ethoxy-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate (407)(1.0 g, 3.65 mmol) in MeOH/H₂O (15 mL, 9:1 by volume) was added LiOH·H₂O(0.15 g, 3.65 mmol) at 0° C. and stirred for 16 h at r.t. The progressof the reaction was monitored by TLC. After completion, the reactionmixture was concentrated under reduced pressure to get a crude residue.The residue was diluted with water (10 mL) and washed with ethylacetate. The aqueous layer was separated, acidified with 1N HCl andextracted with EtOAc (2×50 mL). The combined organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto get 380 mg (42% yield) of3-(2-(tert-butoxycarbonyl)hydrazineyl)-2,2-dimethyl-3-oxopropanoic acid(408) as an off-white solid. LC-MS: m z 245.40 [(M−H)⁻]; R_(t): 1.15min; 57.91% purity.

tert-butyl2-(3-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate(409): To a stirred solution of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (16) (0.3 g, 0.56 mmol),3-(2-(tert-butoxycarbonyl)hydrazineyl)-2,2-dimethyl-3-oxopropanoic acid(408) (0.14 g, 0.56 mmol) in DMF (2 mL) was added triethylamine (0.23mL, 1.68 mmol) followed by T₃P (0.53 mL, 0.84 mmol, 50% solution inEtOAc) at RT. The resultant reaction mixture was stirred at rt for 2 h.The progress of the reaction was monitored by TLC. After completion ofreaction, the reaction mixture was quenched with H₂O (10 mL) andextracted with EtOAc (2×25 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to get 198mg (52% yield) of tert-butyl2-(3-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate(409) as an off-white solid. LC-MS: m z 664.42 [(M+H)⁺]; R_(t): 1.80min; 88.01% purity.

N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydrazineyl-2,2-dimethyl-3-oxopropanamide(410): To a stirred solution of tert-butyl2-(3-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2,2-dimethyl-3-oxopropanoyl)hydrazine-1-carboxylate(409) (0.21 g, 0.32 mmol) in DCM (2 mL) at 0° C., TFA (1 mL) was addedunder nitrogen atmosphere The resultant reaction mixture was allowed tostir at rt for 8 h. The progress of the reaction was monitored by TLC.After completion of reaction, the reaction mixture was concentratedunder reduced pressure to get a crude residue. The crude compound waspurified by RP-Prep-HPLC to afford 33 mg (19% yield) ofN-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydrazineyl-2,2-dimethyl-3-oxopropanamide(410) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.98 (s, 1H),8.16 (d, J=8.4 Hz, 1H), 7.79 (d, J=11.0 Hz, 1H), 7.32 (s, 1H), 6.52 (s,1H), 5.53 (q, J=6.5 Hz, 1H), 5.43 (s, 2H), 5.19 (q, J=19.6 Hz, 2H), 4.27(s, 2H), 3.19-3.09 (m, 2H), 2.40 (s, 3H), 2.16-2.07 (m, 2H), 1.92-1.82(m, 2H), 1.37 (s, 6H), 0.88 (t, J=7.3 Hz, 3H). LC-MS (method 6): m/z564.29 [(M+H)⁺]; R_(t): 1.74 min; 93.90% purity, HP-LC (method 6):R_(t): 3.30 min; 93.05% purity.

3-(2-((E)-1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazinyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide&3-(2-((Z)-1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide(411): To a stirred solutionN-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydrazineyl-2,2-dimethyl-3-oxopropanamide(410) (60 mg, 0.11 mmol) in ethanol was added1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(391) (45 mg, 0.11 mmol) and catalytic amount of acetic acid and thereaction mixture was stirred at 80° C. for 6 h under nitrogen atmosphereThe progress of the reaction was monitored by TLC and LCMS. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby SFC purification to afford 10 mg (10% Yield) of3-(2-((E)-1-(4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazinyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide& 3-(2-((Z)-1-(4-((17-azido-3,6,9,12,15pentaoxaheptadecyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide(411) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (s, 1H),8.40 (d, J=7.5 Hz, 0.11H), 8.11 (d, J=8.2 Hz, 0.73H), 7.72 (q, J=11.1Hz, 1.27H), 7.54 (d, J=8.6 Hz, 1.6H), 7.30 (s, 0.24H), 7.19 (s, 0.82H),6.93 (t, J=10.8 Hz, 0.48H), 6.50 (t, J=17.0 Hz, 2.6H), 5.50-5.39 (m,3.25H), 5.05 (d, J=19.1 Hz, 0.7H), 4.79 (d, J=19.0 Hz, 0.7H), 4.07-4.02(m, 2H), 3.77 (t, J=4.4 Hz, 2H), 3.64-3.50 (m, 18H), 3.37 (t, J=4.9 Hz,2H), 2.93 (q, J=16.3 Hz, 2H), 2.36 (s, 3H), 2.16 (s, 3H), 1.92-1.87 (m,4H), 1.43 (d, J=22.7 Hz, 6H), 0.94 (t, J=7.3 Hz, 3H). LC-MS (method 14):m/z 971.38 [(M+H)⁺]; R_(t): 3.92 min; 99.69% purity, HP-LC (method 14):R_(t): 6.47 min; 99.84% purity. (411) is a mixture of E and Z isomers.

Example 68: Synthesis of3-(2-((E)-1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide&3-(2-((Z)-1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide(412)

To a stirred solution ofN-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydrazineyl-2,2-dimethyl-3-oxopropanamide(410) (120 mg, 0.21 mmol) in ethanol was added1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethan-1-one(397) (147 mg, 0.21 mmol) and catalytic amount of acetic acid and thereaction mixture was stirred at 80° C. for 16 h under nitrogenatmosphere The progress of the reaction was monitored by TLC and LCMS.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound waspurified by SFC purification to afford 25 mg (10% Yield) of3-(2-((E)-1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide&3-(2-((Z)-1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)ethylidene)hydrazineyl)-N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethyl-3-oxopropanamide(412) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): (10.30 (s,0.75H), 10.17 (s, 0.15H), 8.40 (d, J=8.1 Hz, 0.15H), 8.11 (d, J=8.2 Hz,0.77H), 7.75 (t, J=8.9 Hz, 1H), 7.69 (d, J=8.4 Hz, 0.34H), 7.54 (d,J=8.6 Hz, 1.58H), 7.31 (d, J=8.2 Hz, 0.22H), 7.19 (s, 0.79H), 6.95 (d,J=8.1 Hz, 0.34H), 6.50 (t, J=17.3 Hz, 2.47H), 5.51-5.40 (m, J=11.0 Hz,3.4H), 5.05 (d, J=19.0 Hz, 1H), 4.79 (d, J=19.0 Hz, 1H), 4.07-4.02 (m,J=6.3 Hz, 2H), 3.77 (t, J=4.3 Hz, 2H), 3.60-3.49 (m, J=4.6 Hz, 44H),3.00-2.91 (m, J=15.5 Hz, 2H), 2.36 (s, 3H), 2.16 (s, 3.23H), 1.92-1.88(m, J=10.4 Hz, 3.79H), 1.43 (d, J=22.6 Hz, 6H), 0.94 (t, J=7.2 Hz, 3H).LC-MS (method 1): m/z 1235.51 [(M+H)⁺]; R_(t): 1.87 min; 95.04% purity,HP-LC (method 1): R_(t): 4.29 min; 95.11% purity. (412) is a mixture ofE and Z isomers.

Example 69:(Z,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(415)

1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(414): To a stirred solution of17-azido-3,6,9,12,15-pentaoxaheptadecylmethanesulfonate (389) (0.4 g,1.04 mmol) in acetonitrile (20 mL) was added potassium carbonate (216mg, 1.56 mmol) followed by 1-(2-hydroxyphenyl)ethan-1-one (413) (140 mg,1.04 mmol) under nitrogen atm at rt. The resultant reaction mixture wasstirred at 70° C. for 16 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wascooled to 0° C. and concentrated under reduced pressure to get crudecompound. The crude compound was dissolved in water (30 mL) andextracted with EtOAc (2×30 mL). The combined organic layer was washedwith brine solution (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (60-120mesh) eluting with 3% MeOH-DCM to get 350 mg (79% yield) of1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(414) of as a colorless gum. ¹H NMR (400 MHz, CDCl₃): δ 7.77 (dd, J=8.0,2.0 Hz, 1H), 7.48-7.42 (m, 1H), 7.04-7.00 (m, 1H), 7.00-6.96 (m, 1H),4.25 (t, J=4.8 Hz, 2H), 3.93 (t, J=2.0 Hz, 2H), 3.80-3.64 (m, 18H),3.42-3.39 (m, 2H), 2.67 (s, 3H).

(S,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(415): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385) (140 mg, 0.30 mmol) in ethanol (4 mL) was added triethylamine(0.08 mL, 0.60 mmol) and catalytic amount of AcOH followed by1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl) oxy) phenyl)ethan-1-one(414) (128 mg, 0.30 mmol) under nitrogen atmosphere at rt. The resultantreaction mixture was stirred at 80° C. for 16 h. The progress of thereaction was monitored by TLC. After the completion of startingmaterial, reaction mixture was cooled to 0° C., concentrated underreduced pressure to get crude compound. The crude compound was purifiedby SFC purification to afford 7.7 mg (3% Yield) of(S,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide (415) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ10.89 (s, 0.61H), 10.65 (s, 0.30H), 8.14-8.09 (m, 1H), 7.61-7.47 (m,2H), 7.37 (t, J=7.6 Hz, 1H), 7.28 (s, 1H), 7.10 (d, J=8 Hz, 1H), 6.98(t, J=7.2 Hz, 1H), 6.49 (s, 1H), 5.42-5.02 (m, 6H), 4.18 (s, 2H), 4.17(s, 2H), 3.77-3.49 (m, 18H), 3.37-3.32 (m, 2H), 3.12-3.10 (m, 2H),2.33-2.25 (m, 3H), 1.90-1.82 (m, 2H), 1.31-1.23 (m, 4H), 0.87 (t, J=7.2Hz, 3H). LC-MS (method 18): m/z 872.27 [(M+H)]+; R_(t): 2.21 min; 95.05%purity, HP-LC (method 18): R_(t): 6.43 min; 95.55% purity.

Example 70: Synthesis of(S,E)-N′-(1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(418)

1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(417): To a stirred solution of17-azido-3,6,9,12,15-pentaoxaheptadecylmethanesulfonate (389) (0.29 g,0.75 mmol) and 1-(3-hydroxyphenyl)ethan-1-one (416) (92 mg, 0.68 mmol)in acetonitrile (5 mL) was added potassium carbonate (0.2 g, 1.50 mmol).The resultant reaction mixture was heated at 80° C. for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure and the residue obtained was dissolved in water (50 mL),extracted with EtOAc (2×50 mL). The combined organic layer was washedwith brine solution (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (100-200mesh) eluting with 50% EtOAc in pet ether to get 0.2 g (62% yield) of1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(417) as a pale brown gum. LC-MS: m z 448.42 [(M+Na)⁺]; R_(t): 1.75 min;98.26% purity.

(S,E)-N′-(1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(418): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385) (150 mg, 0.32 mmol) in ethanol was added1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(417) (137 mg, 0.32 mmol) and catalytic amount of AcOH at r.t. Thereaction mass heated at 80° C. for 16 h under nitrogen atmosphere Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was purified by SFCpurification to afford 14 mg (5% yield) of(S,E)-N′-(1-(3-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(418) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.99 (s,0.68H), 10.76 (s, 0.25H), 8.12 (d, J=9.2 Hz, 1H), 7.62-7.32 (m, 5H),7.28 (s, 1H), 7.02-7.00 (m, 1.3H), 6.50 (s, 1H), 5.44 (d, J=14.7 Hz,3.38H), 5.30 (s, 2H), 5.04 (s, 0.55H), 4.12 (d, J=4.3 Hz, 2H), 3.74 (d,J=3.8 Hz, 2H), 3.58-3.32 (m, 20H), 3.11 (d, J=7.9 Hz, 2.27H), 2.32 (q,J=4.7 Hz, 3H), 1.87 (t, J=7.7 Hz, 2H), 1.25 (t, J=7.2 Hz, 3.59H), 0.88(t, J=7.3 Hz, 3H). LC-MS (method 16): m/z 872.66 [(M+H)⁺]; R_(t): 1.87min; 95.89% purity, HP-LC (method 16): R_(t): 6.35 min; purity: 95.69%.

Example 71: Synthesis of(S,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide&(S,Z)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(424)

Methyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoate(419): To a stirred solution of(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (1.0 g, 2.55 mmol) in DMF (14 mL) and acetone (4 mL) was addedmethyl 2-bromo-2-methylpropanoate (0.69 g, 3.83 mmol) followed bypotassium carbonate (0.528 g, 3.83 mmol) under Nitrogen atm at rt. Theresultant reaction mixture was stirred at 80° C. for 3 h. The progressof the reaction was monitored by TLC and LCMS. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get a crude mass. Then the crude compound was cooled to 0°C., quenched with ice cold water (30 mL) and stirred for 15 min. Theprecipitated solid was filtered off and triturated with acetone to get0.48 g (38% Yield) of methyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoate(419) as an off-white solid. LC-MS: m z 493.49 [(M+H)⁺]; R_(t): 1.80min; 84.34% purity.

(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoicacid (420): To a stirred solution of methyl(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoate(419) (0.50 g, 1.02 mmol) in THF and H₂O (10 mL, 4:1 by volume) wasadded LiOH·H₂O (0.17 g, 4.08 mmol) at 0° C. The resultant reactionmixture was stirred at rt for 4 h. The progress of the reaction wasmonitored by LCMS and TLC. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude compound was triturated with diethyl ether (20 mL)and acidified with citric acid. The precipitated solid was filtered offand dried under vacuum to get 0.5 g (crude) of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoicacid (420) as an off-white solid. The crude compound was used in thenext step without any further purification. LC-MS: m z 479.46 [(M+H)⁺];R_(t): 1.57 min; 69.40% purity.

tert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoyl)hydrazine-1-carboxylate(421): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoicacid (420) (250 mg, 0.52 mmol) in DMF (5 mL) was added triethylamine(0.22 mL, 1.56 mmol) and T3P (1.32 mL, 2.08 mmol) at 0° C. and thereaction mixture was stirred at 0° C. for 30 min. NH₂NHBoc (172 mg, 1.30mmol) was added to the reaction mixture at 0° C. under Nitrogenatmosphere The resultant reaction mixture was stirred at rt for 16 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with water (20 mL) andextracted with EtOAc (2×20 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to getcrude compound. The crude compound was triturated with diethyl ether (30mL) to get 300 mg (crude) of tert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoyl)hydrazine-1-carboxylate(421) as colorless semi solid. The crude compound was used in the nextstep without any further purification. LC-MS: m z 593.53 [(M+H)⁺];R_(t): 1.70 min; 62.42% purity.

(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(422): A stirred solution of tert-butyl(S)-2-(2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanoyl)hydrazine-1-carboxylate(421) (300 mg, 0.51 mmol) in DCM (3 mL) was cooled to 0° C. 4M HCl in 1,4-dioxane (3 mL) was added to the reaction mixture at 0° C. underNitrogen atmosphere The reaction mixture was stirred at rt for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure to get crude compound. The crude compound was washed withdiethyl ether (20 mL) then dissolved in methanol (25 mL) was addedcarbonate on polymer support resin (150 mg) and the reaction mixture wasstirred at rt for 30 min. The reaction mixture was filtered and thefiltrate was concentrated under reduced pressure to get 200 mg of crudecompound. The crude compound was taken for further purification byRP-prep HPLC to get 11 mg (4% yield) of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(422) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.54 (s, 1H),8.11 (d, J=9.2 Hz, 1H), 7.49-7.46 (m, 2H), 7.28 (s, 1H), 6.49 (s, 1H),5.42 (s, 2H), 5.31 (s, 2H), 4.33 (s, 2H), 3.10 (q, J=7.6 Hz, 2H),1.90-1.86 (m, 2H), 1.52 (s, 6H), 1.29 (t, J=7.2 Hz, 3H), 0.87 (t, J=7.2Hz, 3H). LC-MS (method 3): m/z 493.49 [(M+H)⁺]; R_(t): 1.34 min; 95.58%purity, HP-LC (method 3): R_(t): 3.01 min; Purity: 95.66%.

(S,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide&(S,Z)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(423): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(422) (250 mg, 0.51 mmol) in ethanol (10 mL) was added1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethan-1-one(414) (259 mg, 0.61 mmol) at rt and the reaction mixture was stirred at90° C. for 3 h under nitrogen atmosphere The progress of the reactionwas monitored by TLC and LCMS. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude compound was purified by SFC purification to afford9 mg (2% yield) of(S,E)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide&(S,Z)-N′-(1-(2-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(423) as pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.33 (s,0.2H), 9.34 (s, 0.7H), 8.14 (d, J=9.7 Hz, 0.40H), 8.06 (d, J=9.2 Hz,1H), 7.56 (d, J=7.9 Hz, 0.45H), 7.35-7.17 (m, 3H), 7.06 (d, J=8.4 Hz,0.26H), 6.97 (d, J=8.4 Hz, 1H), 6.88 (q, J=3.0 Hz, 0.7H), 6.72 (t, J=7.4Hz, 0.7H), 6.51 (d, J=3.9 Hz, 1H), 5.43 (d, J=3.6 Hz, 2H), 5.32 (d,J=6.9 Hz, 2H), 4.11 (s, 0.55H), 3.91 (s, 1.5H), 3.59-3.40 (m, 22H), 3.06(t, J=7.7 Hz, 2H), 2.19 (s, 2.3H), 2.05 (s, 0.81H), 1.87-1.80 (m, 2H),1.70 (s, 1.5H), 1.52 (s, 4.5H), 1.26 (t, J=7.6 Hz, 3H), 0.88 (q, J=4.9Hz, 3H). LC-MS (method 7): m/z 900.64 [(M+H)⁺]; R_(t): 4.21, 4.29 min;77.67+21.64% purity; HP-LC (method 7): R_(t): 4.29, 4.36 min;77.45+21.59% purity. Compound (423) is isolated as the mixture of E andZ isomers.

Example 72: Synthesis of(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide (425)

(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide (425): To astirred solution of(S,E)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N′-(1-(4-hydroxyphenyl)-2-methylpropylidene)acetohydrazide(424) (310 mg, 0.51 mmol) in acetonitrile was added potassium carbonate(140 mg, 1.02 mmol) followed by35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontylmethanesulfonate (396) (331 mg, 0.51 mmol) under Nitrogen atmosphere atrt. The resultant reaction mixture was stirred at 80° C. for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure, quenched with ice cold water (30 mL) and extracted with EtOAc(2×50 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to get crude compound. The crudecompound was purified by SFC purification to afford 19 mg (3% yield)(S,E)-N′-(1-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)phenyl)-2-methylpropylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(425) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.55 (d,J=11.2 Hz, 1H), 8.10 (t, J=11.6 Hz, 1H), 7.57-7.40 (m, 1H), 7.28 (s,1H), 7.22-7.04 (m, 4H), 6.49 (s, 1H), 5.43 (s, 2H), 5.31-5.30 (m, 3H),4.81 (s, 1H), 4.15 (t, J=4.4 Hz, 2H), 3.81 (s, 2H), 3.61-3.48 (m, 42H),3.39-3.35 (m, 2H), 3.36-3.32 (m, 2H), 2.89-2.82 (m, 1H), 1.90-1.87 (m,2H), 1.29-1.27 (m, 3H), 1.11-1.04 (m, 6H), 0.88 (t, J=7.2 Hz, 3H). LC-MS(method 3): m/z 1164.79 [(M+H)⁺]; R_(t): 1.95 min; 93.91% purity, HP-LC(method 3): R_(t): 4.42 min; 94.15% purity.

Example 73:(S,E)-N′-(1-(2-((6-azidohexyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(429)

1-(2-((6-bromohexyl)oxy)phenyl)ethan-1-one (427): To a stirred solutionof 1-(2-hydroxyphenyl)ethan-1-one (413) (0.5 g, 3.67 mmol) in DMF (5 mL)was added potassium carbonate (0.76 mg, 5.51 mmol) followed by1,6-dibromohexane (426) (1.79 g, 7.34 mmol) at rt under nitrogenatmosphere. The resultant reaction mixture was stirred at rt for 6 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was quenched with ice cold water(20 mL) and extracted with EtOAc (2×50 mL). The combined organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressurecrude compound. The crude compound was purified by column chromatographyusing silica gel (230-400 mesh) eluting with 20-25% ethyl acetate in Petether to get 0.4 g (36% yield) of1-(2-((6-bromohexyl)oxy)phenyl)ethan-1-one (427) as a colorless gum.LC-MS: m z 299.29 [(M+H)⁺]; R_(t): 2.33 min; 97.08% purity.

1-(2-((6-azidohexyl)oxy)phenyl)ethan-1-one (428): To a stirred solutionof 1-(2-((6-bromohexyl)oxy)phenyl)ethan-1-one (427) (0.4 g, 1.34 mmol)in DMF (4 mL) was added sodium azide (0.17 g, 2.68 mmol) at rt undernitrogen atmosphere. The resultant reaction mixture was stirred at 60°C. for 1 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was quenched withice cold water (20 mL) and extracted with EtOAc (2×30 mL). The combinedorganic layer were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure crude compound. The crude compound was purified bycolumn chromatography using silica gel (230-400 mesh) eluting with50-60% ethyl acetate in Pet ether to get 0.24 g (68% yield) of1-(2-((6-azidohexyl)oxy)phenyl)ethan-1-one (428) as a colorless gum.LC-MS: m z 262.38 [(M+H)⁺]; R_(t): 2.27 min; 99.01% purity.

(S,E)-N′-(1-(2-((6-azidohexyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(429): To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(422) (150 mg, 0.30 mmol) in ethanol (20 mL) was added1-(2-((6-azidohexyl)oxy)phenyl)ethan-1-one (428) (97 mg, 0.37 mmol) atr.t. The reaction mixture was stirred at 90° C. for 2 h under nitrogenatmosphere The progress of the reaction was monitored by TLC and LCMS.After completion of starting material, reaction mixture was concentratedunder reduced pressure to get crude compound. The crude compound waspurified by SFC purification to afford 29 mg (13% yield) of(S,E)-N′-(1-(2-((6-azidohexyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(429) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.26 (s, 1H),8.06 (d, J=9.2 Hz, 1H), 7.30-7.23 (m, 3H), 7.16 (dd, J=9.2, 2.4 Hz, 1H),6.95-6.92 (m, 2H), 6.79 (t, J=7.4 Hz, 1H), 6.51 (s, 1H), 5.44 (s, 2H),5.33 (s, 2H), 3.67 (s, 2H), 3.22 (t, J=6.9 Hz, 2H), 3.06 (q, J=7.9 Hz,2H), 2.17 (s, 3H), 1.90-1.83 (m, 2H), 1.52 (s, 6H), 1.40-1.38 (m, 4H),1.27-1.17 (m, 7H), 0.88 (t, J=7.3 Hz, 3H). LC-MS (method 9): m/z 734.26[(M−H)⁻]; R_(t): 2.67, 2.76 min; 94.86+4.43% purity, HP-LC (method 9):R_(t): 5.05, 5.21 min, 94.40+3.90% purity. LCMS and HPLC analysis showedappearance of minor Z isomer.

Example 74:(S,E)-N′-(1-(2-((6-azidohexyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(430)

To a stirred solution of(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide (385) (300 mg, 0.65 mmol) inethanol (30 mL) was added triethylamine (0.11 mL, 0.78 mmol) andcatalytic amount of AcOH followed by1-(2-((6-azidohexyl)oxy)phenyl)ethan-1-one (428) (169 mg, 0.65 mmol)under nitrogen atm at rt. The resultant reaction mixture was heatedunder reflux for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudecompound was purified by SFC purification to afford 20.9 mg (5% yield)of(S,E)-N′-(1-(2-((6-azidohexyl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(430) as a light yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.43 (s,1H), 8.09-8.07 (m, 1H), 7.55-7.40 (m, 2H), 7.28 (s, 1H), 7.19-7.12 (m,3H), 7.06 (t, J=7.6 Hz, 1H), 6.54-6.50 (m, 1H), 5.42 (s, 2H), 5.33-4.82(m, 4H), 4.03-3.91 (m, 2H), 3.21-3.14 (m, 4H), 2.33-2.21 (m, 3H),1.90-1.82 (m, 2H), 1.68-1.26 (m, 11H), 0.89 (t, J=7.2 Hz, 3H). LC-MS(method 2): m/z 708.56 [(M+H)⁺]; R_(t): 2.06 min; 96.47% purity, HP-LC(method 2): R_(t): 4.86 min; 97.82% purity.

Example 75: Synthesis of(S,E)-N′-(1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(434)

1-(4-(4-bromobutoxy)phenyl)ethan-1-one (432): To a stirred solution of1-(4-hydroxyphenyl)ethan-1-one (390) (5.0 g, 36.72 mmol) in DMF (50 mL)was added 1,4-dibromobutane (431) (4.4 mL, 36.72 mmol) followed bypotassium carbonate (10.15 g, 73.45 mmol) at rt. The resultant reactionmixture was stirred at rt for 6 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, reactionmixture was quenched with water (200 mL) and extracted with EtOAc (2×200mL). The combined organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to get crude compound. The crudecompound was purified by column chromatography using silica gel (230-400mesh) eluting with 15% EtOAc in pet ether to afford 3.6 g (36% yield) of1-(4-(4-bromobutoxy)phenyl)ethan-1-one (432) as a colorless liquid.LC-MS: m z 271.24 [(M+H)⁺]; R_(t): 2.02 min; 99.07% purity.

1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethan-1-one(433): To a stirred solution of17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (388) (2.0 g, 6.51 mmol) inDMF (20 mL) was added NaH (60% dispersion in mineral oil, 0.52 g, 13.01mmol) at 0° C. After stirring for 15 min1-(4-(4-bromobutoxy)phenyl)ethan-1-one (432) (1.76 g, 6.51 mmol) wasadded at 0° C. The resultant reaction mixture was stirred at rt for 3 h.The progress of the reaction was monitored by LCMS. After completion ofstarting material, reaction mixture was quenched with water (100 mL) andextracted with EtOAc (2×100 mL). The combined organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to getcrude compound. The crude compound was purified by RP-prep HPLC to get0.52 g (16% yield) of1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethan-1-one(433) as a pale brown gum. LC-MS: m z 520.24 [(M+Na)⁺]; R_(t): 1.89 min;98.04% purity.

(S,E)-N′-(1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(434): To a stirred solution of1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethan-1-one(433) (214 mg, 0.43 mmol) in ethanol (2 mL) was added(S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385) (200 mg, 0.43 mmol) and triethylamine (0.06 mL, 0.43 mmol)followed by catalytic amount of acetic acid. The reaction mixture wasstirred at 80° C. for 16 h under nitrogen atmosphere The progress of thereaction was monitored by TLC and LCMS. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure toget crude compound. The crude compound was purified by SFC purificationto afford 29 mg (7% yield) of(S,E)-N′-(1-(4-((1-azido-3,6,9,12,15,18-hexaoxadocosan-22-yl)oxy)phenyl)ethylidene)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(434) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.90-10.70(m, 1H), 8.12 (t, J=7.6 Hz, 1H), 7.78 (q, J=10.6 Hz, 2H), 7.56 (q,J=15.8 Hz, 2H), 7.28 (s, 1H), 6.96 (d, J=8.7 Hz, 2H), 6.50 (s, 1H), 5.43(d, J=6.7 Hz, 3H), 5.29 (s, 2H), 5.01 (br. s, 1H), 4.02 (t, J=6.3 Hz,2H), 3.52 (t, J=5.4 Hz, 26H), 3.12-3.09 (m, 2H), 2.29 (d, J=11.5 Hz,3H), 1.89-1.85 (m, 2H), 1.78-1.73 (m, 2H), 1.65 (q, J=7.1 Hz, 2H),1.30-1.22 (m, 3H), 0.88 (t, J=7.3 Hz, 3H). LC-MS (method 27): m/z 944.22[(M+H)⁺]; R_(t): 1.91 min; 96.02% purity, HP-LC (method 27): R_(t): 4.49min; 97.66% purity.

Example 76: Synthesis of(S)—S-(3-((4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)propyl)ethanethioate (327)

Compound (327) was prepared by the procedure outlined in Example 54.

Example 77: Synthesis of(s)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1h-yrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetohydrazide(385)

Compound (385) was prepared by the procedure outlined in Example 64.

Example 78:N-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-Dioxo-2,3,9,10,13,15-Hexahydro-1H,12H-Benzo[De]Pyrano[3′,4′:6,7]Indolizino[1,2-b]Quinolin-1-Yl)-3-Hydrazineyl-2,2-Dimethyl-3-Oxopropanamide(410)

Compound (410) was prepared by the procedure and characterized asoutlined in Example 67.

Example 79:(S)-9-(3-((1-Amino-2-methylpropan-2-yl)disulfanyl)propoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(331)

Compound (331) was prepared was prepared and characterized as outlinedin Example 54.

Example 80:(S)-9-(3-aminopropoxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(105)

Compound (105) was prepared and characterized by the procedure outlinedin Example 15.

Example 81: Synthesis of(s)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-methylpropanehydrazide(422)

Compound (422) was prepared and characterized by the procedure give inthe Example 71.

Example 82: Synthesis ofN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-mercaptoacetamide(452)

Methyl 2-(tritylthio)acetate (449): The mixture of methyl2-mercaptoacetate (362) (1.0 g, 9.42 mmol) and triphenylmethyl chloride(2.62 g, 9.40 mmol) in toluene (10 mL) was stirred at 110° C. for 2 h.The progress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was cooled to rt and evaporatedunder reduced pressure to get a crude residue. The resulting residue wasstirred with methanol (200 mL) and the precipitated solid was filteredoff, washed with methanol (100 mL) and dried under vacuum to affordmethyl 2-(tritylthio)acetate (449) (2.6 g, 79%) as an off-white solid.¹H NMR (400 MHz, CDCl₃): δ 7.43-7.40, (m, 6H), 7.31-7.20 (m, 9H), 3.58(s, 3H), 2.98 (s, 2H).

2-(tritylthio)acetic acid (450): A Suspension of methyl2-(tritylthio)acetate (449) (2.4 g, 6.89 mmol) and 10% potassiumhydroxide in methanol (38.6 mL, 68.87 mmol) was stirred for 4 h at rtand the progress of the reaction was monitored by TLC. After completionof starting material, the reaction mixture was diluted with 50% methanolin water (100 mL) and the resulting mixture was acidified with 3Naqueous HCl (pH-6) at 0° C. The resulting mixture was extracted withchloroform (2×100 mL). The combined organic extract was dried overanhydrous Na₂SO₄, filtered and evaporated under reduced pressure toafford 1.99 g (87% yield) of 2-(tritylthio)acetic acid (450) as anoff-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.34-7.29 (m, 12H),7.24-7.20 (m, 3H), 2.56 (s, 2H).

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-(tritylthio)acetamide(451): To the stirred suspension of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (0.05 g, 0.094 mmol), 2-(tritylthio)acetic acid(450) (0.031 g, 0.094 mmol) and PyBOP (0.073 g, 0.141 mmol) inN,N-dimethylformamide (1.0 mL), N,N-diisopropylethylamine (0.036 g,0.282 mmol) was added at r.t and the reaction mixture was stirred at rtfor 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was diluted withice cold water (10 mL) and stirred for 10 min. The precipitated solidwas filtered off, dried under vacuum to get crude compound. The obtainedcrude compound was purified by Davisil grade silica gel columnchromatography using 5% MeOH in DCM to afford 0.05 g (71% yield) ofN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-(tritylthio)acetamide(451) as an off-white solid. LC-MS: m z 752.41 [(M+H)⁺]; R_(t): 2.42min; 93.08% purity.

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-mercaptoacetamide(452): At 0° C. mixture of TFA/TIPS/water (95:2.5:2.5) (1 mL) was addedtoN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-(tritylthio)acetamide(451) (0.045 g, 0.06 mmol) and the reaction mixture was stirred at rtfor 30 min. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was cooled to 0°C. and diluted with ice cold water (10 mL) and stirred for 10 min. Theprecipitated solid was filtered off, washed with diethyl ether (10 mL),n-pentane (10 mL) and finally dried under vacuum to afford 0.024 g (80%yield) ofN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-mercaptoacetamide(452) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.60 (d, J=8.6Hz, 1H), 7.81 (d, J=10.9 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.55 (m,1H), 5.43 (s, 2H), 5.26 (q, J=21.0 Hz, 2H), 3.16 (m, 4H), 2.86 (t, J=8.1Hz, 1H), 2.41 (s, 3H), 2.16 (m, J=5.5 Hz, 2H), 1.86 (m, 2H), 0.87 (t,J=7.3 Hz, 3H). LC-MS (method 27): m/z 510.12 [(M+H)⁺]; R_(t): 1.61 min;97.06% purity, HP-LC (method 27): R_(t): 3.67 min; 98.03% purity.

Example 83: Synthesis of2-((1-amino-2-methylpropan-2-yl)disulfaneyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide2,2,2-trifluoroacetic acid salt (454)

2-((1-amino-2-methylpropan-2-yl)disulfaneyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide2,2,2-trifluoroacetic acid salt (454): To a stirred solution oftert-butyl(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)disulfaneyl)-2-methylpropyl)carbamate(453) (0.02 g, 0.028 mmol) in DCM (2 mL), TFA (0.5 mL) was added at 0°C. and the reaction mixture was stirred at r.t for 2 h. The progress ofthe reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was evaporated under reduced pressure toget a crude residue. The resulting residue was washed with diethyl ether(3×10 mL), n-pentane (10 mL) and dried under vacuum to afford 0.019 g(93% yield) of2-((1-amino-2-methylpropan-2-yl)disulfaneyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide2,2,2-trifluoroacetic acid salt (454) as a yellow solid. ¹H NMR (400MHz, DMSO-d₆): δ 8.78 (d, J=8.5 Hz, 1H), 7.85-7.82 (m, 4H), 7.33 (s,1H), 6.55 (s, 1H), 5.57-5.56 (m, 1H), 5.43 (s, 2H), 5.33 (d, J=19.1 Hz,1H), 5.22 (d, J=18.9 Hz, 1H), 3.58-3.51 (m, 2H), 3.20-3.10 (m, 2H), 3.00(d, J=4.9 Hz, 2H), 2.42 (s, 3H), 2.16 (q, J=8.0 Hz, 2H), 1.90-1.83 (m,2H), 1.29 (d, J=3.3 Hz, 6H), 0.87 (t, J=7.2 Hz, 3H). LC-MS (method 29):m/z 613.12 [(M+H)⁺]; R_(t): 1.44 min; 95.87% purity, HP-LC (method 29):R_(t): 3.44 min; 95.01% purity.

Example 84: Synthesis of(1S,9S)-1-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione2,2,2-trifluoroacetate (377)

Compound (377) was prepared and characterized by the procedure outlinedin Example 62.

Example 85:(S)-9-((4-((2-((1-amino-2-methylpropan-2-yl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(380)

Compound (380) was prepared and characterized by the procedure outlinedin Example 63.

Example 86:2-(4-aminophenyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(458)

Methyl 2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxyacetate (455):methyl 2-(4-aminophenyl)-2-hydroxyacetate (94) (100 mg, 0.55 mmol) andBoc-anhydride (0.13 mL, 0.55 mmol) were mixed and irradiated inmicrowave at 80° C. for 1 h. The progress of the reaction was monitoredby TLC. After completion of starting material, reaction mixture wastaken up in dichloromethane and concentrated under reduced pressure toget crude compound. The crude compound was purified by columnchromatography using silica gel (100-200 mesh) eluting with 25-30% ethylacetate in pet ether to get 90 mg (58% yield) of methyl2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxyacetate (455) as awhite solid. LC-MS: m z 280.28 [(M−H)⁻]; R_(t): 1.62 min; 99.23% purity.

2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxyacetic acid (456): Toa stirred solution of methyl2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxy acetate (455) (90 mg,0.32 mmol) in THF/H₂O (5.5 mL, 10:1 by volume) was added LiOH·H₂O (54mg, 1.28 mmol) at rt. The resultant reaction mixture was stirred at rtfor 2 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure and water (15 mL) was added followed by acidificationwith saturated citric acid solution and extracted with EtOAc (2×20 mL).The combined organic layer was washed with brine solution (20 mL) anddried over anhydrous Na₂SO₄ and concentrated under reduced pressure toget 70 mg (82% yield) of2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxy acetic acid (456) asa pale yellow gum. LC-MS: m z 266.33 [(M−H)⁻]; R_(t): 1.61 min; 97.30%purity.

tert-butyl(4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)carbamate(457): To a stirred solution of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethane sulfonate (16) (60 mg, 0.11 mmol) in DMF (5 mL) was added2-(4-((tert-butoxycarbonyl)amino)phenyl)-2-hydroxy acetic acid (456) (29mg, 0.11 mmol), PyBOP (88 mg, 0.17 mmol) and DIPEA (0.06 mL 0.33 mmol)at rt. The resultant reaction mixture was stirred at r.t for 2 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was quenched with ice water (10 mL)and the precipitated solid was filtered off, dried under vacuum to get70 mg (91% yield) of tert-butyl(4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)carbamate(457) as a pale brown solid. LC-MS: m z 685.71, 685.66 [(M+H)⁺]; R_(t):2.18, 2.26 min; 31.51+34.50% purity.

2-(4-aminophenyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(458): To a stirred solution of tert-butyl(4-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-hydroxy-2-oxoethyl)phenyl)carbamate(457) (60 mg, 0.09 mmol) in 1,4-dioxane (4 mL) was added 4M HCl in1,4-Dioxane (0.2 mL) at 0° C. The resultant reaction mixture was stirredat rt for 3 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, reaction mixture was concentrated underreduced pressure to get crude compound. The crude compound was purifiedby RP-preparative HPLC to afford 9 mg (18% yield) of2-(4-aminophenyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(458) (HCl salt) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.77 (br.s, 2H), 8.81-8.70 (m, 1H), 7.80 (d, J=10.8 Hz, 1H), 7.64-7.53 (m, 2H),7.32-7.02 (m, 4H), 6.57 (s, 1H), 5.55-5.41 (m, 3H), 5.30-5.10 (m, 3H),3.17-3.12 (m, 2H), 2.40 (s, 3H), 2.18-2.09 (m, 2H), 1.92-1.81 (m, 2H),0.88 (t, J=7.2 Hz, 3H). LC-MS (method 27): m z 585.38 [(M+H)⁺]; R_(t):1.46, 1.48 min; 36.62+60.56% purity, HP-LC (method 27): R_(t): 3.90,3.93 min; Purity: 40.30+57.94%.

Example 87: Synthesis of(S)-9-((4-((2-((2-aminoethyl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(463) (TFA salt)

tert-butyl(2-mercaptoethyl)carbamate (339): To the stirred solution of2-aminoethane-1-thiol (1.0 g, 12.96 mmol) in acetonitrile(10 mL)/water(5 mL), NaHCO₃ (2.18 g, 25.92 mmol) was added. Thendi-tert-butyldicarbonate (5.66 g, 25.92 mmol) was added slowly at 0° C.and the reaction mixture was stirred at room temperature for 4 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was diluted with water (20 mL) andextracted with ethyl acetate (2×100 mL). The combined organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure toget crude compound. The obtained crude compound was purified by silicagel column chromatography using 5% methanol in DCM to afford 600 mg (26%yield) of tert-butyl (2-mercaptoethyl) carbamate (339) as a colorlessgum. ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.02 (s, 1H), 3.44 (q, J=6.0 Hz,2H), 2.79 (t, J=6.4 Hz, 2H), 2.67-2.62 (m, 1H), 1.49-1.44 (m, 9H).

tert-butyl (2-((2-aminoethyl)disulfaneyl)ethyl)carbamate (460): To thestirred solution of tert-butyl(2-mercaptoethyl)carbamate (339) (500 mg,2.68 mmol) in methanol(10 mL) in2-(pyridin-2-yldisulfaneyl)ethan-1-amine (459) (570 mg, 3.22 mmol) wasadded at 0° C. and the reaction mixture was stirred at room temperaturefor 4 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was concentratedunder reduced pressure to get crude compound. The obtained crude waspurified by davisil grade silica gel column chromatography using 4%methanol in dichloromethane to afford 500 mg (70% yield) of tert-butyl(2-((2-aminoethyl)disulfaneyl)ethyl)carbamate (460) as a pale yellowsemi solid. LC-MS: m z 253.16 [(M+H)⁺]; R_(t): 1.18 min; 70.05% purity.

tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(461): To a stirred solution of tert-butyl(2-((2-aminoethyl)disulfaneyl)ethyl)carbamate (460) (692 mg, 2.74 mmol)in N,N-dimethylformamide (15 mL) was added N,N-diisopropylethylamine(0.95 mL, 5.48 mmol) at 0° C. followed by 2,4,6-trichloro-1,3,5-triazine(374) (500 mg, 2.74 mmol). The reaction mixture was stirred at roomtemperature for 1 h. The progress of the reaction was monitored by TLC.After completion of starting material, the reaction mixture was dilutedwith ice cold water (20 mL) and extracted with ethyl acetate (3×30 mL).The combined organic extract was dried over anhydrous Na₂SO₄ andevaporated under reduced pressure to get crude compound. The obtainedcrude compound was purified by trituration with diethylether to afford400 mg (37% yield) of tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(461) as off-white solid. LC-MS: m z 398.17 [(M−H)⁻]; R_(t): 2.22 min;71.78% purity.

tert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(462): To a stirred solution of tert-butyl(2-((2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(461) (408 mg, 1.02 mmol) in DMF (10 mL) was added DIPEA (0.35 mL, 2.04mmol) and(S)-4,11-diethyl-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(34) (400 mg, 1.02 mmol) slowly at 0° C. and the reaction mixture washeated at 60° C. for 3 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure and quenched with ice cold water (30mL). The precipitated solid was filtered off and washed with diethylether to get 400 mg (52% yield) oftert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(462) as an off-white solid. The crude compound was used in the nextstep without any further purification. LC-MS: m/z 756.11 [(M+H)⁺];R_(t): 2.03 min; 86.47% purity.

(S)-9-((4-((2-((2-aminoethyl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(463) (TFA salt): To a stirred solution oftert-butyl(S)-(2-((2-((4-chloro-6-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-1,3,5-triazin-2-yl)amino)ethyl)disulfaneyl)ethyl)carbamate(462) (100 mg, 0.13 mmol) in dichloromethane (5 mL) was added2,2,2-trifluoroacetic acid (1 mL) at 0° C. and the reaction mixture wasstirred at room temperature for 2 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, the reactionmixture was evaporated under reduced pressure to get crude compound. Thecrude compound was purified by RP-Prep HPLC to afford 10.4 mg (11%yield) of(S)-9-((4-((2-((2-aminoethyl)disulfaneyl)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione(463) (TFA salt) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ8.92-8.72 (m, 1H), 8.25-8.15 (m, 2H), 7.83-7.77 (m, 4H), 7.34 (s, 1H),6.53 (s, 1H), 5.44-5.32 (m, 4H), 3.58-3.40 (m, 2H), 3.20-3.08 (m, 4H),2.92-2.76 (m, 4H), 1.88-1.86 (m, 2H), 1.31-1.26 (m, 3H), 0.90 (t, J=7.2Hz, 3H). LC-MS (method 29): m/z 656.23 [(M+H)⁺]; R_(t): 1.44 min; 97.03%purity, HP-LC (method 29): R_(t): 3.07 min; 97.45% purity.

Example 88: Synthesis of Folic Acid Precursors

Folic acid conjugate precursors suitable for preparing a folate receptortargeting NDC disclosed herein can be prepared according to one of thefollowing synthetic protocols. As the folic acid conjugate precursorscomprise a terminal azide group, they are suitable for attaching to ananoparticle functionalized with alkyne moieties (e.g., DBCO), usingclick chemistry.

Synthesis of(S)-16-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oicacid (606)

Preparation of compound 600: Compound 599 (160 g, 512 mmol) wasdissolved in TFAA (800 mL) at 25° C. and stirred under a nitrogenatmosphere in the dark for 5 hrs. The solvent was then removed at 50° C.in vacuo to give the crude product. The crude product was trituratedwith MTBE (750 mL) for 60 min and then filtered to afford compound 600(203 g, crude) as a solid, which was used in next step without furtherpurification. LC-MS: ¹H NMR: (400 MHz, CDCl₃) δ 12.74 (br s, 1H), 8.88(s, 1H), 7.97-8.05 (m, 2H), 7.66-7.74 (m, 2H), 5.26 (s, 1H).

Preparation of Compound 602: TBTU (238 g, 740 mmol) and DIPEA (95.7 g,740 mmol) were added to a solution of compound 601 (225 g, 529 mmol) inDMF (2.25 L). After 30 min stirring at 20° C.,2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (Reagent A; 121 g,555 mmol) was added and the mixture was stirred at 50° C. for 12 hrs.Two reaction mixtures were combined and worked up, and the residue wasdiluted with H₂O (3 L) and extracted with ethyl acetate (1500 mL×3). Thecombined organic layers were washed with brine (800 mL×3), dried overNa₂SO₄, filtered and concentrated under reduced pressure, and purifiedby column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to1/1) to afford compound 602 (590 g) as an oil. ¹H NMR: (400 MHz, CDCl₃)(7.76-7.78 (m, 2H), 7.63-7.60 (m, 2H), 7.41-7.27 (m, 4H), 6.43 (s, 1H),5.70 (s, 1H), 4.42-4.38 (m, 2H), 4.24-4.23 (m, 2H), 3.63-3.36 (m, 16H),2.28-2.18 (m, 3H), 1.98-1.96 (m, 1H), 1.48 (s, 9H).

Preparation of Compound 603: N-ethylethanamine (1.27 kg, 17.4 mol) wasadded to a solution of compound 602 (435 g, 695 mmol) in DCM (4.35 L)and the mixture was stirred at 25° C. 3 hrs. The solvent was thenremoved at room temperature in vacuo, and the residue was purified byflash column chromatography (DCM/MeOH=100/1 to 1/1) to afford compound603 (245 g) as an oil. ¹H NMR: (400 MHz, CDCl₃) δ 6.55 (s, 1H),3.67-3.30 (m, 17H), 2.34-2.30 (m, 2H), 2.10-2.06 (m, 1H), 1.87 (s, 2H),1.77-1.73 (m, 1H), 1.44 (s, 9H).

Preparation of compound 604: TBTU (119 g, 372 mmol) and DIEA (160 g,1.24 mol) were added to a solution of compound 600 (101 g, 248 mmol) inDMF (900 mL) and the mixture was stirred for 30 minutes. Then compound603 (100 g, 248 mmol) in DMF (100 mL) was added. The mixture was stirredat 25° C. for 12 hrs. Two reaction mixtures were combined andconcentrated and the residue was diluted with H₂O (2.5 L) and extractedwith ethyl acetate (1 L x 5). The combined organic layers were washedwith brine (600 mL×3), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to afford compound 4 (420 g, crude) as a solid,which was used in next step without further purification.

Preparation of compound 605: K₂CO₃ (585 g, 4.23 mol) was added to asolution of compound 604 (420 g, 529 mmol) in THF (4.2 mL) and H₂O (500mL) and the mixture was stirred at 60° C. for 0.5 hr. The reactionmixture was concentrated under reduced pressure to remove THF and theresidue was diluted with H₂O (500 mL) and adjusted the pH to 3 with HCl(M=1), filtered and concentrated under reduced pressure to affordcompound 605 (260 g, crude) as a solid, which was used directly withoutpurification.

Preparation of compound 606: Trifluoroacetic acid (2.12 kg, 18.6 mol)was added in one portion to a mixture of compound 605 (260 g, 373 mmol)in CH₂Cl₂ (2.6 L) at 20° C. under nitrogen, and the mixture was stirredat 20° C. for 5 hrs. The reaction mixture was concentrated under reducedpressure and purified by HPLC (column: Agela DuraShell C18 250*80 mm*10um; mobile phase: [water (10 mM NH₄HCO3)-MeOH]; B %: 5%-40%,20 min) togive afford compound 606 (52.5 g, 81.82 mmol, 21.96% yield) as a solid.(M+H) 642.80; IR: 2107 (N₃ Bond).

Synthesis of(S)-38-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (472)

tert-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465): To a stirred solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (450mg, 0.881 mmol), in DCM (20 mL) at 0° C. was added TEA (0.36 mL, 2.64mmol), EDC (218 mg, 1.14 mmol), HOBT (154 mg, 1.14 mmol) and tert-butyl(2-aminoethyl)carbamate (464) (124 mg, 0.881 mmol) at 0° C. Theresulting reaction mixture was stirred at rt for 16 h. The progress ofthe reaction was monitored by TLC. After consumption of startingmaterial, the reaction mixture was extracted with DCM and water and theorganic layer was dried over Na₂SO₄, and evaporated under vacuum. Thenit was purified by flash chromatography and dried under vacuum to get0.450 g (69% yield) of tert-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465) as a liquid. ¹H NMR (400 MHz, DMSO-d₆): 7.83 (t, 1H), 6.75 (t,1H), 3.61-3.31 (m, 38H), 3.02-2.97 (t, 4H), 2.28 (t, 2H), 1.37 (s, 9H).

N-(2-Aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466):A solution of tert-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465) (350 mg, 0.462 mmol) in DCM was cooled to 0° C. and TFA was addedto it by dropwise, and the reaction mixture was then stirred at RT for16h. The progress of the reaction was monitored by TLC. Afterconsumption of starting material, reaction mixture was concentratedunder reduced pressure and azeotroped with DCM (3 times) to get crudecompound. The crude compound was purified by flash chromatographyeluting with 5% MeOH in DCM to get 0.250 g (97% yield) ofN-(2-aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466) as a liquid. ¹H NMR (400 MHz, DMSO-d₆): 8.02 (t, 1H), 7.73 (t,2H), 3.71-3.26 (m, 40H), 2.86 (t, 2H), 2.35 (t, 2H).

Tert-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467): To a stirred solution of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoicacid (170 mg, 0.4 mmol), in DMF (5 mL) was added DIPEA (0.174 mL, 1.0mmol), PyBOP (416 mg, 0.8 mmol) andN-(2-aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466) (331 mg, 0.6 mmol) at 0° C. The resulting reaction mixture wasstirred at rt for 16 h. The progress of the reaction was monitored byTLC. After consumption of starting material, reaction mixture wasevaporated under vacuum at low temperature. Then it was purified underflash chromatography eluting with 5% MeOH in DCM to get 0.350 g (yield91%) of tert-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467) as a liquid. MH⁺962, retention time 1.81 min.

Tert-butyl(S)-38-amino-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468): To a stirred solution of tert-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467) (350 mg, 3.65 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at rt. The resultant reaction mixture was stirred at rt for 3h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure to get 250 mg of Tert-butyl(S)-38-amino-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468) crude compound. The crude compound was used for the next stepwithout purification. MH⁺739, retention time 1.50 min.

Tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470): To a stirred solution of4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzoicacid (469) (100 mg, 0.245 mmol), in DMF (5 mL) was added DIPEA (0.107mL, 0.613 mmol), PyBOP (254 mg, 0.49 mmol) and tert-butyl(S)-38-amino-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468) (271 mg, 0.368 mmol) at 0° C. The resultant reaction mixture wasstirred at rt for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum under low temperature. Then it was purify underflash chromatography eluting with 10% MeOH in DCM to get 180 mg (yield65%) of tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470) as a solid. MH⁺1129, retention time 2.61 min.

(S)-38-(4-(N-((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (471): To a stirred solution of tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470) (180 mg, 0.16 mmol) in DCM was added TFA (0.123 mL, 1.59 mmol) atRT. The resultant reaction mixture was stirred at RT for 16h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, reaction mixture was concentrated under reducedpressure and azeotrope with DCM (3 times) to get 100 mg crude compound(471). That material was used for the next step without purification.MH⁺1073, retention time 2.34 min.

(S)-38-(4-(((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (472): To a stirred solution of(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (471) (80 mg, 0.071 mmol) in DMF (3 mL) at 0° C. was added Aq.NH₃(dissolve in DMF) (0.01 mL, 0.71 mmol). The resultant reaction mixturewas stirred at rt for 6 h. After completion of starting material,reaction mixture was concentrated under reduced pressure to get crudecompound. The crude material was purified by RP- prep-HPLC to get thedesired compound (472) 15 mg (21% yield) as a solid. ¹H NMR (400 MHz,DMSO-d₆): 8.62 (S, 1H), 8.01 (d, 1 H), 7.98(t,1H),7.64 (d, 2H), 6.64 (d,2H), 4.47 (d, 2H), 4.21 (t, 1H), 3.68-3.35 (m, 38H), 3.07 (t, 4H),2.32-2.11 (t, 6H), 1.86 (t, 1H). LCMS: MH⁺977, retention time 1.96 min.

LCMS Method: Column- YMC TRIART C₁₈ (33×2.1 mm, 3u); (mobile phase: 95%[0.1% HCOOH in water] and 5% [0.1% HCOOH in CH₃CN] held for 0.50 minthen to 1% [0.1% HCOOH in water] and 99% [0.1% HCOOH in CH₃CN] in 3.0min, held this composition up to 4.00 min and finally back to initialcondition in 4.10 min, held for 4.50 min). Flow rate- 1.0 ml/min.

Synthesis of(S)-4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)-N-(5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-1-hydroxy-1,5-dioxo-115-pentan-2-yl)benzamide(479)

Tert-butyl(S)-4-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(473): To a stirred solution of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoicacid (500 mg, 01.17 mmol), in DMF (10 mL) was added DIPEA (0.51 mL, 2.94mmol), PyBOP (1123 mg, 2.35 mmol) and tert-butylpiperazine-1-carboxylate (328 mg, 1.76 mmol) at 0° C. The resultantreaction mixture was stirred at rt for 16 h. The progress of thereaction was monitored by TLC. After completion of starting material,reaction mixture was evaporated under vacuum under low temperature toget crude compound. The crude compound was purified by flashchromatography eluting with 5% MeOH in DCM to get 0.5 g (71% yield) oftert-butyl(S)-4-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(473) as an off white solid. LCMS: MH⁺594, retention time 1.97 min.

Tert-butyl(S)-4-(4-amino-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(474): To a stirred solution of tert-butyl(S)-4-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(473) (500 mg, 0.842 mmol) in DMF (5 ml), 30% piperidine in DMF (1 ml)was added at rt. The resultant reaction mixture was stirred at rt for 3h. The progress of the reaction was monitored by TLC. After completionof starting material, reaction mixture was concentrated under reducedpressure. Then it was purify under flash chromatography eluting with 10%MeOH in DCM to get 0.3 g (95% yield) of tert-butyl(S)-4-(4-amino-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(474) as a sticky solid. LCMS: MH⁺372, retention time 3.03 min.

Tert-butyl(S)-4-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(475): To a stirred solution of4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzoicacid (469) (240 mg, 0.588 mmol), in DMF (10 mL) was added DIPEA (0.25mL, 1.47 mmol), PyBOP (611 mg, 1.17 mmol) and tert-butyl(S)-4-(4-amino-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(474) (320 mg, 0.882 mmol) at 0° C. The resultant reaction mixture wasstirred at rt for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum at low temperature to get the crude compound.The crude compound was purified by flash chromatography eluting with 10%MeOH in DCM to get 0.3 g (67% yield) of tert-butyl(S)-4-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(475) as an off white solid. LCMS: MH⁺762, retention time 1.76 min.

Tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-oxo-5-(piperazin-1-yl)pentanoate(476): To a stirred solution of tert-butyl(S)-4-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(tert-butoxy)-5-oxopentanoyl)piperazine-1-carboxylate(475) (300 mg, 0.394 mmol) in DCM was added TFA (stock solution 1 mL TFAin 9 mL DCM) (0.1 mL, 1.18 mmol) at 0° C. The resultant reaction mixturewas stirred at RT for 3h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasconcentrated under reduced pressure and azeotrope with DCM to get 0.2 g(76% yield) of tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-oxo-5-(piperazin-1-yl)pentanoate(476) crude compound and used for the next step without purification.LCMS: MH⁺662, retention time 2.61 min.

Tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-5-oxopentanoate(477): To a stirred solution of Azido-PEG9-Acid (100 mg, 0.196 mmol), inDMF (5 mL) was added DIPEA (0.08 mL, 0.489 mmol), PyBOP (172 mg, 0.333mmol) and tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-oxo-5-(piperazin-1-yl)pentanoate(476) (194 mg, 0.294 mmol) at 0° C. The resultant reaction mixture wasstirred at rt for 16 h. The progress of the reaction was monitored byTLC. After completion of starting material, reaction mixture wasevaporated under vacuum at low temperature to get crude compound. Thecrude compound was purified by flash chromatography eluting with 10%MeOH in DCM to get 0.015 g (67% yield) of tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-5-oxopentanoate(477) as a brown solid. LCMS: MH⁺1155, retention time 2.73 min.

(S)-4-(N-((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)-N-(5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-1-hydroxy-1,5-dioxo-115-pentan-2-yl)benzamide(478): To a stirred solution of tert-butyl(S)-2-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-5-oxopentanoate(477) (150 mg, 0.13 mmol) in DCM was added TFA (0.1 mL, 1.3 mmol) at RT.The resultant reaction mixture was stirred at RT for 16h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, reaction mixture was concentrated under reduced pressure andazeotrope with DCM to get 0.013 g of(S)-4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)-N-(5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-1-hydroxy-1,5-dioxo-115-pentan-2-yl)benzamide(478) crude as an off white solid and used for the next step withoutpurification. LCMS: MH⁺1099, retention time 2.23 min.

(S)-4-(((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)-N-(5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-1-hydroxy-1,5-dioxo-115-pentan-2-yl)benzamide(479): To a stirred solution of(S)-4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)-N-(5-(4-(1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oyl)piperazin-1-yl)-1-hydroxy-1,5-dioxo-115-pentan-2-yl)benzamide(478) (130 mg, 0.121 mmol) in DMF(3 mL) at 0° C. was added Aq.NH3 (0.02mL, 1.21 mmol). The resultant reaction mixture was stirred at rt for 6h. After completion of starting material, reaction mixture wasconcentrated under reduced pressure to get crude compound. The crudematerial was purified by RP-prep HPLC. After RP-Prep HPLC purification35 mg (yield 21%) of (479)) was obtained as a sticky yellow solid. ¹HNMR (400 MHz, DMSO-d₆): 12.5 (s, 1H), 11.42 (s. 1H), 8.64 (s. 1H),8,12(d, H), 7.64 (t, 2H), 6.96 (t, 1H), 6.64 (d, 2H), 4.74 (d, 2H), 4.29(m, 1H), 3.62-3.31 (m, 38H), 2.62-2.32 (m, 8H), 2.66 (t, 2H), 1.97 U,2H), 1.94 (t, 211), LCMS: MH⁺1003, retention time 1.21 min.

LCMS Method: Column- ZORBAX C₁₈ (50×4.6 mm, 5u), (mobile phase: from 90%[10 mM NH40 Ac in water] and 10% [CH3CN] to 70% [10 mM NH40Ac in water]and 30% [CH3CN] in 1.5 min, further to 10% [10 mM NH40Ac in water] and90% [CH₃CN] in 3.0 min, held this mobile phase composition up to 4.0 minand finally back to initial condition in 5.0 min). Flow=1.2 ml/min.

Example 90: Synthesis of Nanoparticles

Aqueous synthesis methodology can be used for the preparation andfunctionalization of ultrasmall nanoparticles of the present disclosure.For example, methodology based on the procedures outlined in WO2016/179260 A1 and WO 2018/213851 A1 (the contents of which areincorporated herein by reference in their entireties) may be used.

NDCs comprising the nanoparticle (also referred as C′Dot) can beprepared with DBCO functionalization as outlined in the flow chartpresented in FIG. 2 .

For example, a fluorescent compound such as, but not limited to Cy5, canbe functionalized with a maleimide group, to provide amaleimide-functionalized fluorescent compound that has a net positivecharge. This can be conjugated with a thiol-silane, such as(3-mercaptopropyl)trimethoxysilane (MPTMS) to produce a silanefunctionalized fluorescent compound such as Cy5-silane. The conjugationmay be performed in dimethyl sulfoxide (DMSO) in a glovebox under inertatmosphere overnight (16-24 hours) and at room temperature (18-25° C.).

On the following day, the next step of the synthesis can be performed ina suitable chamber, such as a glass flask, container, or reactor, andcan involve stirring deionized water with a pH of around 8.5-10.5 whichcan be achieved using an aqueous solution of ammonium hydroxide of pH7.5-8.5. A silica precursor, such as a tetraalkyl orthosilicate, e.g.,tetramethyl orthosilicate (TMOS) can then be added into the reactionchamber under vigorous stirring at room temperature, followed byimmediately adding the silane-functionalized fluorescent compound, e.g.,Cy5-silane. The reaction can be left stirring at room temperatureovernight (1-48 hours), to provide silica cores encapsulating thefluorescent compound, e.g., Cy5 dye.

The following day, a PEG-silane can be added into the reaction understirring at room temperature to coat the silica core with PEG molecules,and the reaction can be left stirring for 1-48 hours. This step may befollowed by heating between 75-85° C. for 1-48 hours. The reaction canthen be cooled down to room temperature and purified (e.g., includingsterile filtration to remove aggregates formed as side-product of thereaction, and bacteria if any present). Further functionalization of thenanoparticle may then be performed.

Functionalization of Nanoparticles

A nanoparticle prepared using a method disclosed herein may be furtherfunctionalized, e.g., using a method outlined in FIG. 2 or 3 , or inScheme 82 below. For example, (3-cyclopentadienylpropyl)triethoxysilane(“diene-silane”) can be used to functionalize a nanoparticle (e.g.,C′Dot) with cyclopentadiene groups, then DBCO-PEG-maleimide can bereacted with the diene-functionalized nanoparticle to provide aDBCO-functionalized nanoparticle.

For example, Cy5-C′Dot (which may be prepared using a method describedherein) was diluted with deionized water to a desired concentration,typically between 15 to 30 μM, in a round-bottom flask with a stir bar.(3-Cyclopentadienylpropyl)triethoxysilane (cyclopentadiene) was firstdiluted 100x in DMSO and then added into the reaction with stirring, toreach a desired particle to cyclopentadiene molar ratio. After overnightreaction, 10× PBS was added into the reaction to achieve a finalconcentration of 1× PBS. Next, a DBCO-maleimide precursor (e.g.,DBCO-PEG4-maleimide) was dissolved in DMSO and added into the reactionto reach a desired particle to DBCO molar ratio. After mixing for about30 min to 1 hour, the reaction mixture was heated to 80° C. whilestirring overnight. The reaction solution was then concentrated andpurified using gel permeation chromatography (GPC) to obtain diene-basedDBCO-C′Dot. For example, a the concentration of freshly preparednanoparticles may be adjusted, and a cyclopentadiene-functionalizedsilane can be added, and stirred at room temperature for about 1-48hours, followed by the addition of a DBCO-maleimide, with furtherstirring at room temperature for an additional 1-48 hours, followed bypurification, and a step of heating the reaction to 75-85° C. for 16-24hours, followed by a final step of purification, to yield a DBCOfunctionalized C′Dot (referred as C′Dot or DBCO-C′Dot).

The purification may be performed based on the principle of sizeseparation. Aggregates and free small molecules having molecular weightdifferent than that of the pegylated nanoparticles are separated usinggel permeation chromatography columns (GPC) or Tangential FlowFiltration (TFF) system. Two different membranes, 300 kDa, and 50 kDacut-off sizes were employed for the removal of large aggregates and freesmall molecules respectively. Both GPC and TFF systems can be used totransfer the aqueous medium to water, saline etc. Purified DBCO-C′Dot indeionized water can be sterile filtered again and the quality control(QC) steps can be performed, followed by storage in refrigerator at 2-8°C.

Without wishing to be bound by theory, it is believed that the neutralcharge of the cyclopentadiene groups averts hydrolysis of the amidebonds in the linkage, that can be accelerated by other types ofprecursors (e.g., when using amine-silanes instead of diene-silanes, theprimary amine groups can cause hydrolysis). Thus, the NDCs producedusing this method are highly stable (see, e.g., comparison in FIGS.33A-33B). Additionally, using diene-functionalized nanoparticles (e.g.,cyclopentadiene-functionalized nanoparticles) in the preparation of NDCSgreatly diminishes the self-condensation of silane during the reaction,and improves the stability, size homogeneity, reaction yield, and purityof the functionalized nanoparticles, relative to other methods (e.g.,using amine-silanes).

Example 91: Synthesis of NDCs

NDCs of the present disclosure comprising the nanoparticle (alsoreferred as C′Dot), targeting ligand such as Folic Acid and linker-drugconjugates can be prepared as outlined in the flow chart presented inFIG. 3 . By adjusting the amount of targeting ligand precursor used inthe functionalization step, a desired number of targeting ligands pernanoparticle can be achieved. For example, nanoparticles of the presentdisclosure may be functionalized to contain about 10 to about 20targeting ligands, e.g., about 10, about 11, about 12, about 13, about14, or about 15 targeting ligands. Similarly, by adjusting the amount ofpayload-linker conjugate precursor used in the functionalization step, adesired number of payload moieties per nanoparticle can be achieved. Forexample, nanoparticles of the present disclosure may be functionalizedto contain about 10 to about 40 payload moieties, e.g., about 20, about21, about 22, about 23, about 24, or about 25 payload moieties.

DBCO-C′Dot (referred as C′Dot in FIG. 3 ) was diluted using deionizedwater to a concentration of 15-45 μM. After the temperature ofDBCO-C′Dot solution was around 18-25° C., folate receptor (FR)-targetingligand precursor such as, folic acid (FA) functionalized with an azide(e.g., a compound prepared according to Example 88, such as Compound606) was dissolved in DMSO (0.021 M) and was then added into thereaction with stirring at room temperature, providing a C′Dotfunctionalized with FA via the DBCO group on the surface. The reactionratio between DBCO-C′Dot and FA was kept from 1:5 to 1:30, and thesolution was stirred for 16-24 hours at temperature of 18-25° C.FR-targeting ligand addition is followed by sterile filtration,purification and QC testing to yield FA-C′Dot (referred as C′Dotintermediate in FIG. 3 ), and can be stored in a refrigerator at 2-8° C.FA-C′Dot comprises a portion of DBCO groups that are available forfurther click-reactions, e.g., g with molecules with azidefunctionality. It will be understood that the folate-targeting ligand(e.g., folic acid) can be conjugated to the nanoparticle afterconjugation with, e.g., a payload-linker conjugate.

The volume of the FR-targeting ligand conjugation reaction can rangefrom 5 mL to 30 L, and the concentration of DBCO-C′Dot can range from 15to 45 μM. The following parameters are given for a typical reactionvolume of 600 mL and a DBCO-C′Dot concentration of 25 μM. The ratio ofDBCO-C′Dot to FR-targeting ligands was precisely controlled to obtainthe desired number of FR-targeting ligands per particle, and typicallycan range from 1:5 to 1:30. For a typical ratio of 1:12,folate-PEG-azide was dissolved in DMSO to a concentration of 0.021 M,and 8.571 mL of the folate-PEG-azide/DMSO solution was added into thereaction. After stirring overnight at room temperature, the reactionmixture was either purified to obtain FA-C′Dot or continue directly tonext conjugation step if the purity of FA-C′Dot is no less than 95%. Theconversion rate of FR-targeting ligand is typically higher than 95%.

The number of Folic Acid groups attached onto each FA-C′Dot wascharacterized by UV-Vis, and a representative UV-Vis absorbance spectrumis shown in FIG. 4 . The number of DBCO groups on each C′Dot can becalculated using the extinction coefficient of C′Dot and DBCO groups

FA-C′Dots were diluted using deionized water to a concentration of 15-45μM. After the FA-C′Dot solution temperature reached around 18-25° C.,the linker-payload conjugate precursor, e.g., exatecan-cathepsin Bcleavable drug linker with azide functionality (linker-drug conjugatesof Formula (I)-(XII)) dissolved in DMSO (0.04 M) was added into thereaction under stirring at room temperature. This step functionalizedthe FA-C′Dot with the linker-drug conjugate via the available DBCOgroups on the surface. The reaction ratio between FA-C′Dot andlinker-drug conjugate was kept around 1:10-1:50 and the solution wasstirred for 16-24 hours. The addition of linker-drug conjugate wasfollowed by sterile filtration, and purification. FA-CDC (also referredas NDC) in deionized water is QC tested, and stored in refrigerator at2-8° C.

The volume of the cleavable exatecan conjugation reaction can range from5 mL to 30 L, and the concentration of FA-C′Dot can range from 15 to 45μM. The following parameters are given for a typical reaction volume of600 mL and a FA-C′Dot concentration of 25 μM. The ratio of FA-C′Dot tocleavable exatecan was precisely controlled to obtain the desired numberof cleavable exatecan per particle, and typically can range from 1:10 to1:60. For a typical ratio of 1:40, cleavable exatecan was dissolved inDMSO to a concentration of 0.04 M, and 15 mL of the cleavableexatecan/DMSO solution was added into the reaction. After stirringovernight at room temperature, the reaction mixture was purified toobtain FA-CDC. It will be understood that this method can be used forconjugating any of the drug-linker conjugate precursors disclosed hereinto a carrier particle.

The number of Exatecan payloads attached onto each NDC, e.g., Folic Acid(FA)-functionalized drug-linker conjugated C′Dot (FA-CDC), may becharacterized by UV-Vis. A representative UV-Vis absorbance spectrum isshown in FIG. 5 . The number of Exatecan payloads on each C′Dot can becalculated using the extinction coefficient of C′Dot and Exatecan at 360nm after the subtraction of the absorption of Folic Acid at the samewavelength.

As stated above, a nanoparticle may be functionalized with a targetingligand and a payload-linker conjugate in any order (e.g., the protocoloutlined above for functionalizing the nanoparticle with exatecan may becarried out prior to the protocol for conjugating the targeting ligand).Additionally, these methods can be used to conjugate a nanoparticle toany desired payload or targeting ligand, e.g., via any suitable linker.For example, any of the payload-linker conjugate precursors or targetingligand precursors described herein, including in the above Examples, maybe conjugated to a nanoparticle using these methods.

Example 92: Particle Size Determination

The average diameter of NDCs can be measured by any suitable methods,such as, but not limited to Fluorescence Correlation Spectroscopy (FCS)(FIG. 6 ) and Gel Permeation Chromatography (GPC) (FIG. 7 ).

FCS detects the fluorescence fluctuation resulted from particlediffusion through the focal spot on the objective. Particle diffusioninformation is then extracted from the autocorrelation of signalintensity fluctuations, from which the average hydrodynamic particlesize can be obtained by fitting the autocorrelation curve using asingle-modal FCS correlation function. The average hydrodynamic diameterof NDC was about 6 nm to about 7 nm (FIG. 6 ).

GPC is a type of molecular sieving chromatography, where the separationmechanism is based on the size of the analyte (here NDC's). The elutiontime of NDC is compared to a series of proteins with varying molecularweight. The results suggest that the elution time of NDC's is comparableto that of protein standards with molecular weight between 158 kDa and44 kDa, consistent with the particle size average hydrodynamic size ofabout 6.4 nm (FIG. 7 ).

Example 93: Purity Analysis

The purity of NDCs was analyzed using reversed phase HPLC (RP-HPLC).RP-HPLC is coupled to a photodiode array detector, using a commerciallyavailable Waters Xbridge Peptide BEH C18 column. RP-HPLC separatesmolecules with different polarities and is suitable as an analyticalmethod for NDCs because of its ultrasmall sub-10 nm particle size.

Using RP-HPLC, the nanoparticles are well separated from aggregates andother chemical moieties such as targeting ligands that arenon-covalently associated with the nanoparticles and degraded products.Different chemical moieties are identified based on their elution timeand unique UV/Vis spectra. The photodiode array detector collects UV-Visspectra from 210 to 800 nm, and impurities of interest are measured at330 nm. A representative chromatogram shown for the NDCs in FIG. 8 ,suggests that the purity of NDCs of the present disclosure is higherthan 99.0%.

Example 94: Cell Viability Assay

The following example describes an in vitro assay that was conducted todetermine the cell viability of exemplary payloads.

Method: Human KB cells (ATCC® CCL-17™) were cultured in folic acid freerpmi-1640 medium (ThermoFisher, GIBCO) for at least 7 days before thestudy. The cells were seeded in opaque 96-well plates and allowed toattach overnight (cell density was carefully controlled to be 3,000cells per well at the time of assay). After that, cells were treatedwith drug analogs (suspended in the same RPMI-1640 medium) at aconcentration of zero to 1,000 nM. After 7 days, cell viability wasassessed using the CellTiter-Glo® 2.0 assay (Promega) according to themanufacturer's instructions. The percent of viable cells at eachconcentration was calculated by normalizing the luminescence values tothe untreated control. The half-maximal inhibitory concentration (IC₅₀)of the payloads were plotted and fitted by using the Prism 8 software(GraphPad). Table 2 provides the IC₅₀ of the payloads of the presentdisclosure (‘+’ indicates 1-9.9 nm; ‘++’ indicates 10-100 nm).

TABLE 2 IC₅₀ of Payloads IC₅₀ values (diluted in RPMI Payload No.(Example No.) medium) (nM) 385 (77) ++ 327 (76) ++ 105 (80) + 454 (83)++ 422 (81) ++ 452 (82) ++ 377 (84) ++ 380 (85) ++ 458 (86) + 463 (87)++ 410 (78) ++ 331 (79) ++

Example 95: Drug-Release Assays

NDCs of the present disclosure comprise a linker-payload conjugate, thatmay comprise a cathepsin-B (Cat-B) cleavable linker, redox-sensitive(otherwise known as redox-responsive) linker, or a pH-sensitive linker.Several types of payloads including, e.g., a cytotoxic drug moleculedescribed herein, such as (but not limited) to SN-38, analogs of SN-38,exatecan and analogs of exatecan can be conjugated to a linker, toprovide one of the linker-payload conjugates of the present disclosure.In order to screen the linker-payload conjugates towards development ofNDCs, the linker-payload conjugates can be attached to a nanoparticle(e.g., the silica-based nanoparticle platform referred to as C′Dot), andthe drug-releasing profile and the stability of linker-drug conjugateson the nanoparticle (e.g., C′Dot) can be tested.

To assess the drug releasing profile of exemplary linker-drugconjugates, the linker-drug conjugates were each conjugated to aDBCO-functionalized ultrasmall silica nanoparticle via a click chemistryreaction between the azide groups on the linker-drug conjugateprecursors and the dibenzocyclooctyne (DBCO) groups of thenanoparticles. The resulting drug-nanoparticle-conjugates were thenincubated under the desired releasing conditions for release kineticstests.

Both SN-38 and exatecan exhibit absorption maxima at wavelength around360 nm (FIG. 9A and FIG. 9B), and such a signal can be used to trace thepayloads in high-performance liquid chromatography (HPLC) for releasingand stability studies. The amount of released drugs vs non-releaseddrugs was measured using reverse phase HPLC by analyzing the area undercurve (AUC) (FIG. 10A and FIG. 10B).

General Method: A Waters Xbridge Peptide BEH C18 column with 4.6 mm×50mm dimensions, a particle size of 5 μm, and a pore size of 300 A wasused (part number 186003622). Acetonitrile (VWR HiPerSolv Chromanorm,UHPLC Grade) was used as received without further preparation, 0.010%trifluoroacetic acid in deionized water was prepared by adding 1 mL oftrifluoroacetic acid (HPLC grade, Millipore-Sigma) into 999 mL 18.2MΩ·cm deionized water that was generated using an IQ7000 Milliporedeionized water system and passed through a 0.2 μm filter before use.The seal wash used for the system was composed of 90% 18.2 MΩ·cmdeionized water and 10% methanol (HPLC grade, VWR). The injection needlewas washed using a mixture of 25 vol % 18.2 MΩ·cm deionized water, 25vol % acetonitrile, 25 vol % methanol, and 25 vol % 2-propanol. Sampleswere prepared in a concentration range of 0.25 to 2 μM and the injectionvolume ranges from 60 μL to 10 μL, respectively. Higher sampleconcentration can be used if detector signal is low. Vials used for allinjections are fresh Waters Total Recovery vials with screw caps thathave pre-slit PTFE septa (part number 186000385C).

Method for Protease-cleavable and Redox-responsive Linkers: Before anysample injections were started, the PDA lamp was turned on and allowedto warm up for at least 30 minutes. The system and column wereequilibrated with 95% 0.01% TFA in deionized water, 5% acetonitrile forat least 10 minutes at a flow rate of 1.0 mL/min after the PDA lamp hadwarmed up. Two blank injections, with injection volumes of 10 μLcontaining only 18.2 MΩ·cm deionized water, were performed before theinjection of any samples for analysis. The gradient used to eluteprotease-cleavable and redox-sensitive linkers conjugated to hybridsilica nanoparticles, and their components began at 95% 0.01% TFA indeionized water and 5% acetonitrile and linearly changed to 15% 0.01%TFA in deionized water, 85% acetonitrile over 8 minutes. Acetonitrilecomposition was increased to 95% over an additional minute and held at95% for an additional 2 minutes to ensure that any strongly retainedcompounds are eluted. The composition of the solvent was then changedback to the starting composition of the gradient over an additionalminute and allowed to equilibrate for 3 minutes before another injectionbegan. Between sample injections a blank injection was run to ensurethat no carryover occurred.

Method for pH-sensitive Linkers: Before any sample injections werestarted, the PDA lamp was turned on and allowed to warm up for at least30 minutes. A gradient of pure deionized water and acetonitrile wasused, all other consumables including column and vials were the same asfor the method to purify Cat-B sensitive and redox-responsive linkerselaborated on above. Briefly the gradient composition began at 95%deionized water and 5% acetonitrile at a flow rate of 1.0 mL/min (notchanged throughout the method). The composition was changed from thisstarting composition to 15% deionized water and 85% acetonitrile over a12-minute period, during which the retained drug-linkers, released drug,free drug linker, and any other impurities eluted. At the 12-minute markthe composition was rapidly changed to 5% deionized water and 95%acetonitrile over the course of one minute. This composition was heldfor an addition 2 minutes to ensure any late eluting impurities werefully washed off the column. The composition was then changed back tothe starting composition and equilibration for the next injection wasstarted. For the analysis of all cleaving data, Empower 3 ApexTrackintegration was used to determine peak areas for all relevantcomponents.

For a typical Cat-B protease cleaving study, 2 μL, 0.33 μg/μL of Cat-B(sigma Aldrich) was first added with 300 μL of activation buffer (25 mMMES, 5 mM DTT, pH 5.0), forming 2.2 μg/mL of Cat-B. The mixture was keptat room temperature for 15 min before use. After activation, 100 μL of 2μM drug-nanoparticle-conjugate was mixed with 100 μL of activated Cat-B.The mixture was then transferred to 37° C. To monitor the cleavingkinetics, at selected post-incubation time points (e.g., 2, 4,24 h), 10μL of mixture was sampled and injected in HPLC (TFA/acetonitrile). Forthe analysis of cleaving data, Empower 3 ApexTrack integration was usedto determine peak areas for all relevant components.

For a typical redox-responsive cleaving study, 10 μL of 10 μMdrug-nanoparticle-conjugate was diluted into 89 μL of PBS. Then, 1 μL of1 M microbiology grade dithiothreitol was added to the PBS solution. Themixture was then transferred to 37° C. To monitor the cleaving kinetics,at selected post-incubation time points (e.g., 2, 4, 24 h), 20 μL ofmixture was sampled and injected in HPLC (TFA/acetonitrile). For theanalysis of cleaving data, Empower 3 ApexTrack integration was used todetermine peak areas for all relevant components. Two of widely usedbuffer solutions in this study were 1×PBS (pH=7.4), and sodium acetate(pH 5.5).

For a typical pH-sensitive cleaving study, 100 μL of 2 μMdrug-nanoparticle-conjugate was mixed with 100 μL of buffer with variedpH values (from <5 to >7). The mixture was then transferred to 37° C. Tomonitor the cleaving kinetics, at selected post-incubation time points(e.g., 2, 4, 24 h), 10 μL of mixture was sampled and injected in HPLC(deionized water/acetonitrile). For the analysis of cleaving data,Empower 3 ApexTrack integration was used to determine peak areas for allrelevant components. Two of widely used buffer solutions in this studywere 1×PBS (pH=7.4), and sodium acetate (pH 5.5).

The percentage of drug released during 24 hrs time period is presentedin Table 3. Additionally, the HPLC chromatograph of three representativeCat-B cleavable drug-linkers is depicted in FIGS. 11A-I IC respectively.FIG. 11A depicts the reverse-phase HPLC chromatograph of an exemplaryNDC prepared using Compound 89 from Example 10, at different time pointsafter incubation with cathepsin-B enzyme. FIG. 11B depicts thereverse-phase HPLC chromatograph of an exemplary NDC prepared usingCompound 158 from Example 25, at different time points after incubationwith cathepsin-B enzyme. FIG. 11C depicts the reverse-phase HPLCchromatograph of an exemplary NDC prepared using Compound 202 fromExample 33, at different time points after incubation with cathepsin-Benzyme.

The time for half of the payloads to be released, i.e. T_(1/2), underthe specific experimental condition was analyzed by fitting and isdepicted in FIGS. 4A-4C respectively. FIG. 12A depicts the T_(1/2) as2.9 hours for an exemplary NDC prepared using Compound 89 from Example10. FIG. 12B depicts the T_(1/2) as 2.6 hours for an exemplary NDCprepared using Compound 158 from Example 25. FIG. 12C depicts theT_(1/2) as 1.4 hours for an exemplary NDC prepared using Compound 202from Example 33.

Table 3 demonstrates the results of a drug release assay using certainof the linker-payload conjugates disclosed herein. Each of thelinker-payload conjugates tested was stable, as 5% or less of the drugwas released from the linker drug conjugate after 24 hours undernon-cleavage conditions, i.e., when maintained in PBS, human serum ormouse serum. The release of drug from the linker-payload conjugatesafter 24 hours under cleavage conditions are shown in Table 3. (‘+’indicates 5-25% release; ‘++’ indicates 26-50% release; ‘+++’ indicates51-75% release; ‘++++’ indicates 76-100% release).

TABLE 3 Percentage of Payload Released in 24 hours Linker-Payload % ofPayload Release in 24 Hours Prepared According to: Under CleavageConditions Example 9 ++++ Example 10 +++ Example 11 +++ Example 12 +Example 13-R isomer + Example 13-S isomer + Example 14-R isomer +Example 14-S isomer + Example 15 ++ Example 16 +++ Example 17 ++ Example18 +++ Example 19 + Example 20 + Example 21 + Example 22 + Example 23 +Example 24 + Example 25 +++ Example 26 + Example 27 ++++ Example 28 +++Example 29-Peak 1 +++ Example 29-Peak 2 ++ Example 30 ++ Example 31 +Example 32 + Example 33 ++++ Example 46 + Example 47 + Example 48 +Example 49 + Example 50 + Example 54 + Example 55 + Example 56 + Example57 +++ Example 58 ++++ Example 59 +++ Example 60 +++ Example 61 +Example 64 +++ @ 3 Hrs Example 67 ++++ Example 68 ++++

Stability Test:

To assess the drug releasing profile and stability of exemplarylinker-drug conjugates under non-cleavage conditions, the linker-drugconjugates were first conjugated to the ultrasmall silica nanoparticlevia the click chemistry reaction between the azide groups on thelinker-drug conjugates and the dibenzocyclooctyne (DBCO) groups of thenanoparticles (according to the Examples disclosed herein).

The resulting drug-nanoparticle-conjugates were then incubated inphosphate-buffered saline (PBS) buffer, human and mouse serum at 37° C.for stability tests.

For a typical stability test in PBS buffer, 600 μL of PBS mixture(drug-nanoparticle-conjugate concentration was kept at 2 μM, while thevolume percentage of PBS was kept as 50%) was prepared and kept at 37°C. To monitor the stability of the linker-drug conjugates attached tonanoparticles, at selected post-incubation time points (e.g., 4, 24, 48and 72 h), 10 μL of mixture was sampled and injected in HPLC(TFA/acetonitrile). For the analysis of cleaving data, Empower 3ApexTrack integration is used to determine peak areas for all relevantcomponents.

For a typical stability test in plasma from varied species (e.g., mouse,rat, dog, monkey and human), 600 μL plasma mixture(drug-nanoparticle-conjugate concentration was kept at 2 μM, while thevolume percentage of plasma was kept as 62.5%) was prepared and kept at37° C. To monitor the stability of linker-drug conjugates, at selectedpost-incubation time points (e.g., 4, 24, 48 and 72 h), 80 μL of mixturewas first mixed with 80 μL of cold acetonitrile, and then went through30 min of centrifugation at 10,000 rpm. After removal of the proteins,60 μL of supernatant was carefully sampled and injected in HPLC. ForCathepsin-B and redox-responsive drug-nanoparticle-conjugate,TFA/acetonitrile was used and for pH-sensitivedrug-nanoparticle-conjugate, deionized water/acetonitrile condition wasused. For the analysis of stability data, Empower 3 ApexTrackintegration was used to determine peak areas for all relevantcomponents.

Example 96: In Vitro Flow Cytometry Cell Binding Study

Cell-binding activity of exemplary NDCs (“FA-CDCs”) disclosed herein wastested according to the following protocols. NDCs used were preparedaccording to the Examples disclosed herein.

Cells and Cell Culture: Human KB cell line, SKOV-3 cells and TOV-112cell line were purchased from ATCC. I-GROV1, human ovarian carcinomacell line was purchased from EMD Millipore. Cells were maintained inFolic Acid free RPMI 1640 media/10% FBS, and 1% ofpenicillin/streptomycin, unless otherwise specified.

Cancer cells were cultured in Folic Acid-free medium (RPMI1640,ThermoFisher, GIBCO) for at least one week before the study. Cellbinding studies were performed by incubating 5×105 cells (total of 500uL, 1 Million/mL) in cold PBS (with 1% of BSA) with FA-CDC(concentration: 1 nM) for 60 min at 4° C. (n=3). After that, the cellsuspension was stained with viability kit (LIVE/DEAD™ Fixable VioletDead Cell Stain Kit, Thermo Fisher) for 10-15 min. Then, cells werecentrifuged (2000 rpm, 5 min), washed (2-3 times) using cold PBS (with1% of BSA) before resuspending in PBS (with 1% of BSA). Triplicatesamples were analyzed on a LSRFortessa flow cytometer (BD Biosciences)(Cy5 channel, 633 nm/647 nm, Live/dead cell stain, 405 nm). Results wereprocessed using FlowJo and Prism 7 software (GraphPad).

The competitive binding study (FIG. 13 ) was performed using the NDC ofExample 3. The active targeting of the NDC (FA-CDC) can be fully blockedby incubating with the presence of 1 mM free Folic Acid.

The competitive binding study shows >40-fold enhancement in bindingcapability of the NDC (FA-CDC) when compared with free folic acid,demonstrating the presence of a multivalent effect when conjugatingmultiple folic acid ligands on each ultrasmall C′Dot (FIG. 13A).

The flow cytometry MFI shows >50-fold enhancement in Folate Receptoralpha positive KB cell line when compared to Folate Receptor alphanegative TOV cell line (100 nM, 4° C., 60 min) (FIG. 13B).

These results demonstrates the advantages of conjugating multiple smalltumor-directing ligands on the surface of nanoparticle (C′Dots) forenhancing the active targeting capability using the multivalent effect.The folate receptor targeting can be blocked by competitive binding offree folic acid, such as by incubating with the presence of 1 mM freefolic acid.

The flow cytometry shows comparable folate receptor targeting efficacyof two exemplary NDC (FA-CDC) formulations with varied folic acid liganddensity, in KB cell line. The linker-drug conjugate precursor used toprepare the NDC used in this study is described in Example 33 (Compound202). Blocking in the the blocking group was achieved using 1 mM of freefolic acid. (FIG. 14 ).

The results demonstrated dramatic increase (>300-fold of MFI) in folatereceptor-alpha active targeting when the folic acid ligand density wasincreased from zero to 12 (i.e., 12 folic acid molecules pernanoparticle), while little difference was observed upon furtherincreasing that density to 25 folic acid molecules per nanoparticle.

The flow cytometry shows comparable folate receptor targeting efficacyof three NDCs in KB cell line with varied drug per particle (DPR) (i.e.,number of exatecan molecules per nanoparticle). The blocking groupinvolved blocking receptors with 1 mM of free folic acid. The NDCs withdifferent ratios of exatecan per nanoparticle were prepared usingCompound 202 described in Example 1, and the results of the study areprovided in FIG. 15 . All FA-CDCs comprise between 12 and 22 folic acidmoieties. FA-CDCs with high drug-particle ratio (DPR) comprise between35 and 50 exatecan-linker conjugate groups. FA-CDCs with medium DPRcomprise between 17 and 25 exatecan-linker conjugate. FA-CDCs with lowDPR have between 5 and 10 exatecan-linker conjugate groups.

These results together with the nearly unchanged FCS sizing changes ofthe three NDCs demonstrate the robust surface chemistry and maintainedfolate receptor targeting capability of NDCs disclosed herein, which issurprisingly not perturbed by altering the loading capacity of payload,and demonstrates a significant advantage of the NDCs disclosed hereinover other drug delivery platforms.

Pre-incubating NDCs in human plasma did not negatively affect folatereceptor targeting ability. This study was designed to test the possiblenegative impact of human plasma on the NDCs, such as the formation ofprotein corona. The formation of protein corona and its negative impacton the designed active targeting capability of drug delivery system hasbeen well documented in the literature. The results of this flowcytometry study are depicted in FIG. 16 , which show nearly unchangedfolate receptor targeting efficacy of NDCs at 1 nM, after preincubationwith varied amounts of human plasma for 24 hours. The NDCs were preparedusing the exatecan-payload conjugate precursor of Example 33 (Compound202). The blocking group involved blocking with 1 mM of free folic acid.This study clearly demonstrated that the formation of a protein corona(if any) on the NDC had nearly no negative impact on the in vitrotargeting capability of the NDCs.

Example 97: In vitro Cell Viability Assay

The in vitro cytotoxicity of the NDCs disclosed herein were tested incancer cells. The cancer cells were cultured in folic acid-free medium(RPMI1640, ThermoFisher, GIBCO) for at least one week before the study.Cells were plated in opaque 96-well plates at a density of 3×10³ cellsper well (total of 90 mL) and allowed to attach overnight. The followingday, cells were treated with NDC (FA-CDC) prepared using thelinker-payload conjugate precursor compound 202 (Example 33), at aconcentration range of 0-50 nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 50 nM) by adding 10 mL of 10x stock FA-CDC Solution.

Cells were treated for a pre-defined exposure time (depending on thestudy design, e.g., 4-6 hours, or 7 days). In the case ofshort-exposure-time viability study, cancer cells in each wells werewashed with 100 mL PBS and refreshed with 100 mL of cell medium. Afterwashing, the plates were returned back to 37° C. incubator for 7 daysbefore the viability assay. In the case of 7-day-exposure-time viabilitystudy, no additional washing step was performed. After 7 days, the cellviability was assessed using the CellTiter-Glo2.0 assay (Promega)according to manufacturer's instructions. Data for both viability andproliferation were plotted using Prism7 software (GraphPad).Representative cell viability results of six FA-CDCs with similarsurface density of Folic Acid targeting ligands and drug linkers ispresented in provided in Table 4.

TABLE 4 Representative cell viability results of FA-CDCs with similarsurface density of Folic Acid targeting ligands and linker-drugconjugates. Linker-Payload Conjugate IC50 in KB IC50 Prepared in cellline inSKOV-3 cell Example # Linker Type (++++) nM line (+++) nM 10.Cat-B cleavable 5.2-0.2 10.7 70. pH sensitive   1-0.5 17.9 68. pHsensitive 7.2-0.7 n.t 18. Cat-B cleavable 17.5 n.t. 25. Cat-B cleavable2.2-0.2 0.4 27. Cat-B cleavable  5.2 n.t 28. Cat-B cleavable 72.2 n.t.75. pH sensitive 42.7 n.t. 57. RedOx sensitive  0.6 0.9 58. RedOxsensitive 49.4 n.t. 33. Cat-B cleavable  0.3 0.13 Number of FA ligandsper particle is between 12 and 22; Number of linker-drug conjugates perparticle is between 17 and 25. “n.t” denotes not tested.

Example 98: Two-Dimensional (2D) Confocal Imaging of Exemplary NDC inCancer Cells

A 2D confocal imaging study was carried out to determine the targetingof cells with varying levels of folate-receptor availability using NDCsof the present disclosure. The cells with high folate-receptorexpression (denoted++++) were KB cells. The cells with no FR expression(denoted (—) were TOV-112D cell line. FR-blocked cells were also used.

KB cells were maintained in folic acid free rpMI 1640 media with 10%FBS, 1% penicillin/streptomycin. TOV-112D cells were maintained in 1:1mixture of MCDB 105 medium containing a final concentration of 1.5 g/Lsodium bicarbonate and Medium 199 containing a final concentration of2.2 g/L sodium bicarbonate, supplemented with 15% FBS and 1%penicillin/streptomycin. Cells were trypsinized and seeded in 8-wellLab-Tek chambered coverglass, at 1.0×105 cells per well, and culturedovernight to allow for attachment. Before incubation with FA-CDC, cellswere washed once with folic acid free RPMI 1640 media. FA-CDC was addedinto folic acid free RPMI 1640 media to final concentration of 50 nM.For blocking conditions, folic acid (20 mM stock dissolved in 0.1 MNaOH) was added to final concentration of 0.1 mM and co-incubated withFA-CDC. Cells were incubated with FA-CDC at 37° C. for either 1 hours or24 hours. After incubation, cells were washed three times. To stainlysosomes, LysoTracker Green DND-26 (Thermo Fisher Cat. L7526,ex/em504/511 nm) was added to final concentration of 100 nM in FolicAcid free RPMI 1640 media with 10% FBS, 1% P/S, and incubated at 37° C.for 45 min. Cells were washed once to remove remaining lysotracker dyes.To stain nuclei, Hoechst 33342 solution (Thermo Fisher Cat.62249, 20 mM)was diluted 1:4000 in Folic Acid free RPMI 1640 media with 10% FBS, 1%P/S, and incubated at 37° C. for 10 min. Cells were washed once, andmedia was exchanged to phenol red free RPMI 1640 media for confocalimaging using Nikon spinning disk confocal microscope, 60x objective,405 nm, 488 nm, 640 nm laser lines, exposure time 100 ms for 405channel, 500 ms for 488 channel, and 600 ms for 640 channel.

Results from confocal microscope imaging of FA-CDC in KB(++++) andTOV-112D(—) cell lines at 1 hr time point showed that FA-CDC were mainlypresent at the cell membrane of KB cells, which express high level ofFolate Receptors, but not in blocking conditions or folate negative cellline TOV-112D, suggesting specific binding of FA-CDC to FolateReceptors. After 24 hrs, membrane bound FA-CDC were internalized and theamount of internalized FA-CDC significantly increased as compared with 1hr time point. The internalized FA-CDC were localized in acidicorganelles stained by LysoTracker, indicating that the trafficking ofFA-CDC occurred though the endo-lysosomal pathway. The effect of serumon the binding capability of FA-CDC was also investigated by incubatingFA-CDC particles in media supplemented with 10% FBS overnight prior toincubating them with cells, and no significant difference was observed(data not shown), suggesting that the presence of serum had no impact onthe binding capability of FA-CDC.

The results of this assay are provided in FIG. 17 , and demonstrate thehighly specific active targeting and lysosome trafficking of the NDCs ofthe present disclosure, indicating that once the FA-targeting NDCs bindto cells they become internalized in folate receptor positive celllines, where the payload may be cleaved (e.g., by cathepsin-B) torelease the payload in the cancerous cell.

Example 99: Confocal Imaging of FA-CDC in 3D Tumor Spheroid Model in KBCells

A 3D tumor spheroid model assay was conducted to determine the tumorpenetration of the NDCs disclosed herein. The assay compared anexemplary NDC (prepared according to methods disclosed herein usingexatecan-linker conjugate precursor 202 of Example 33), with apayload-free FA-targeting nanoparticle (prepared according to methodsdisclosed herein with only the FA precursor and without exatecan-payloadconjugate precursor); a folate receptor (FR)-targeting ADC; and thecorresponding payload-free FR-targeting antibody. The FR-targetingantibody was prepared based upon the published sequence of mirvetuximab(provided in U.S. Pat. No. 9,637,547 as huMov19; the contents of whichare incorporated herein by reference in its entirety). The ADC wasprepared with the same antibody and was conjugated to the maytansinoiddrug DM4 (created by Syngene International Ltd.) via a4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sSPDB) linker (based onthe linker used in U.S. Pat. No. 9,637,547). The ADC and antibody wereeach conjugated with Cy5 organic dye, by reaction with Cy5-NHS ester,and were purified by a PD-10 column.

Corning ultra-low attachment surface 96-well spheroid microplates wereutilized in seeding KB cells for having KB spheroids with cell density10,000/well. Single-cell suspensions were generated from trypsinizedmonolayers and diluted to 100,000 cells/mL using RPMI medium (Folic Acidfree). 100 mL of cell suspension were dispensed into each well of amicroplate. The plate was kept in an incubator for 24 hours for cellsforming spheroids. KB cell spheroids can be easily observed bymicroscope with 10× objective.

3D KB spheroids formed after overnight culturing in an ultra-low 96-wellmicroplates. NDC (prepared using exatecan-linker conjugate precursor 202of Example 33), folate-targeted nanoparticles (“FA-C′Dot”), FR-targetedADC, or payload-free FR-targeted antibody were added into wells (n=3)with 50 nM final concentration and incubated for 4 hours at 37° C. Eachtreated KB spheroid and control spheroid were washed with PBS for threetimes and then carefully transferred to a glass bottom 96-well plate(Cellvis) for observation by Nikon AIR-STED confocal microscope, usinglaser line 640 nm, 20 X objective. Z-stacks were acquired by taking2-dimensional images each separated by 1 m in the Z-direction.

Results from the Z-stack confocal microscope imaging of KB tumorspheroid treated with the NDC, FA-C′Dot, FR-targeted ADC, andpayload-free FR-targeted antibody is depicted in FIG. 18 . The resultsshow that the penetration and well diffusion of NDC and FA-C′Dotsthroughout the whole >800 mm of tumor spheroids. In contrast, labeledantibody and ADC merely accumulated around, but not inside of, the tumorspheroids. The ability of the NDCs disclosed herein to achieve efficienttumor penetration is highly advantageous, and shows significantimprovement compared to conventional drug delivery platforms.

Example 100: ⁸⁹Zr Radiolabeling of DFO-FA-CDC and In Vivo Static PET/CTand Biodistribution Studies

A radiolabeling assay was conducted to determine the in vivobiodistribution of the folate receptor-targeting NDCs of the presentdisclosure. The NDCs used for the assay were conjugated with thechelator desferrioxamine (DFO) and then bound with a radionuclide(⁸⁹Zr).

For a typical ⁸⁹Zr labeling, about 1 nmol of DFO-conjugated FA-CDC weremixed with 1 mCi of ⁸⁹Zr-oxalate (produced and provided by University ofWisconsin-Madison Cyclotron group) in HEPES buffer (pH 8) at 37° C. for60 min; final labeling pH was kept at 7-7.5. The labeling yield could bemonitored by using radio instant thin-layer chromatography (iTLC). Anethylenediaminetetraacetic acid (EDTA) challenge procedure was thenintroduced to remove any nonspecifically bound ⁸⁹Zr from the particlesurface. As-synthesized ⁸⁹Zr-DFO-FA-CDC were then purified by using aPD-10 column. The final radiochemical purity was quantified by usingiTLC.

For PET/CT imaging, healthy nude mice (n=3) were i.v.-injected with200-300 μCi (7.4-11.1 MBq)⁸⁹Zr-DFO-FA-CDC. Approximately 5 min prior tothe acquisition of PET/CT images, mice were anesthetized by inhalationof 2% isoflurane/oxygen gas mixture and placed on the scanner bed;anesthesia was maintained using 1% isoflurane/gas mixture. PET/CTimaging was performed in a small-animal PET/CT scanner (InveonmicroPET/microCT) at 1-2, 24, 48, and 72 h post-injection. An energywindow of 350-700 keV and a coincidence timing window of 6 ns were used.Data were sorted into 2D histograms by Fourier rebinning, and transverseimages were reconstructed by filtered back-projection into a 128×128×63(0.72×0.72×1.3 mm3) matrix. The PET/CT imaging data were normalized tocorrect for nonuniformity of response, dead-time count losses, positronbranching ratio, and physical decay to the time of injection; noattenuation, scatter, or partial-volume averaging corrections wereapplied. The counting rates in the reconstructed images were convertedto activity concentrations (percentage injected dose per gram of tissue,% ID/g) by use of a system calibration factor derived from the imagingof a mouse-sized water-equivalent phantom containing ⁸⁹Zr.Region-of-interest (ROI) analyses of the PET data were performed usingIRW software. At 72 h post-injection, organs from each individual mousewere collected, wet-weighted and gamma counted (Automatic Wizard2γ-Counter, PerkinElmer). The uptake of ⁸⁹Zr-DFO-FA-CDC was presented as% ID/g (mean±SD).

The NDCs of the present disclosure enable precise tumor targeting, deeptumor penetration and high tumor killing efficacy. The NDCs can becleared rapidly and efficiently from the body, which reduces thepotential for off-target toxicities and results in an improved safetyprofile. The NDCs disclosed herein can be administered to a subject andcirculate through the blood stream, target the cancer (e.g., tumor),diffuse, penetrate, internalize, and be cleaved to release the payload,killing the cancer cells.

In this study, the renal clearance and biodistribution pattern of FA-CDCwere tested. As shown in FIG. 19A, after the intravenous injection, the⁸⁹Zr-DFO-FA-CDC circulated in the blood stream of healthy nude mouse, asindicated by the high radioactive signal from the heart and artery.Dominant radioactive signal can also be seen from the mouse bladder,demonstrate the renal clearance of the NDC. After 24 h, the majority ofthe injected ⁸⁹Zr-DFO-FA-CDC was cleared out of the mouse body. Thechanges in biodistribution pattern at 2 hour and 24 hour post-injectionis also shown in FIG. 19B. As expected, the NDC can circulate in theblood stream with a dominant renal clearance pathway, whilst avoidingclearance by the mononuclear phagocytic system (MPS) (i.e., liver andspleen).

Example 101: Human KB Tumor Model and In Vivo Efficacy Study

The in vivo efficacy of the NDC was carried out using a human KB tumormouse model. The compared used the following NDCs: NDC A prepared usingthe linker-payload conjugate precursor Compound 342, provided in Example57; NDC B prepared using the linker-payload conjugate precursor Compound87, provided in Example 10; NDC C prepared using the linker-payloadconjugate precursor Compound 158, provided in Example 25; NDC D preparedusing the linker-payload conjugate precursor 202, provided in Example33; NDC E prepared using the linker-payload conjugate precursor Compound418, provided in Example 70; and NDC F prepared using the linker-payloadconjugate precursor Compound 430, provided in Example 74. Each NDC wascompared to a control and free exatecan, and NDCs E and F were comparedto free exatecan and irinotecan (CPT-11).

Human KB cell line was purchased from ATCC and maintained in Folic Acidfree RPMI 1640 media/10% FBS, and 1% of penicillin/streptomycin, unlessotherwise specified. Once the KB cells were cultured to reach anadequate cell count, the cell viability was confirmed by a hemocytometerand trypan blue staining assay. For subcutaneous implantation, eachmouse was injected with KB cells at a density of 2×106 cells/mice at 0.1mL Matrigel/cell dilution volume per injection on the left lower flankof the thigh. Once a subcutaneous tumor volume has reached a palpablesize of 75 to 150 mm³ in a required number of mice for this study, themice was randomized and assigned to each treatment cohort resulting withcomparable tumor volume statistics. Following randomization and studycohort assignment, each dose cohort was treated according to the routesof administration, dosage and schedule.

Two dose levels of each of NDCs B-D were used in the efficacy study (andone dose level for NDCs A, E and F). Tumor volume measurements wereperformed using a calibrated caliper every second day during the dosetreatment period, followed by twice weekly measurements during therecovery period of the in-life phase, and tumor volumes were determinedusing the formula length (mm) x width (mm) x width (mm)×0.50. Bodyweight measurements were performed every second day during the dosetreatment period, followed by twice weekly measurements during therecovery period of the in-life phase. Mice were euthanized when the endpoints of the study reached 1000 mm³. Tumors were harvested and tumorsize was measured. Tumor were surgically excised and snap-frozen forstorage at −80° C. until future analysis.

FIGS. 20A-20F depicts the in vivo tumor growth inhibition studies of thesix folate receptor-targeting NDCs (FA-CDCs) in KB tumor-bearing mice(n=7). The tumor growth charts depicted for the in vivo efficacy studyshows a clear response of tumor growth inhibition in mice treated withNDCs A-D, while mice treated with NDC E or NDC F showed no significantinhibition in tumor growth. Doses for the NDC comprising cathepsin-Benzyme cleavable linkers (such as groups of mice that receivedNDCs B, C,or D), and NDC comprising pH- and di-thiol drug linkers (i.e., groups ofmice that received NDCs A, E, or F) are provided in FIGS. 20A-20F.Control group mice received normal saline follow the same Q3DX3 doseregimen.

Example 102: Activity of NDCs in Drug-Resistant Cell Lines Compared toFree Drug

An assay was carried out using the NDCs disclosed herein to determinetheir activity in drug-resistant cancer cells (specifically,irinotecan-resistant KB cells and extecan-resistant KB cells). The NDCsused in this assay were prepared using the linker-payload conjugateprecursor 202 (from Example 33).

Development of TOP1 Inhibitor-Resistant Folate Receptor Alpha PositiveCancer Cells.

Naïve human KB cell line were purchased from ATCC and maintained infolic acid free RPMI 1640 media/10% FBS, and 1% ofpenicillin/streptomycin. To develop the TOP1 inhibitor resistant KBcells, the cells in flask (50-60% confluence) were repeatedly treatedwith increasing concentration of exatecan, topotecan, SN-38 oririnotecan for over 4 months. The starting TOP1 inhibitor treatmentconcentration was close to the KB cell's IC₉₀ values. After eachtreatment, the cells were carefully washed with fresh RPMI 1640 media,and left to proliferate for an additional 2-3 days until reaching 50-60%confluence. The next round of TOP1 inhibitor treatment was started with2-10× higher TOP1 inhibitor concentration.

Resistant Factor and IC₅₀ Assay.

Both naïve and TOP1 inhibitor resistant KB cell were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO). Cells were plated inopaque 96-well plates at a density of 3×103 cells per well (total of 90μL) and allowed to attach overnight. The following day, cells weretreated with selected TOP1 inhibitors or FA-CDC at suitableconcentration ranges. After exposing the TOP1 inhibitors with both typesof cells for the same period of time, the cell viability was assessedusing the CellTiter-Glo2.0 assay (Promega) according to manufacturer'sinstructions. Data for both viability and proliferation were plottedusing Prism7 software (GraphPad). The resistant factor can be calculatedby using the following equation:

${{Resistant}{factor}} = \frac{{IC}_{50}{of}{resistant}{KB}{cell}}{{IC}_{50}{of}{naive}{KB}{cell}}$

Irinotecan-Resistant KB Cell Line and Potency Test of NDC

FIG. 21A shows the IC₅₀ curves of irinotecan in both naïve and resistantKB cells, which demonstrates the successful development of 5×Irinotecan-resistant KB cells, where IC₅₀ free irinotecan inirinotecan-resistant KB cells was 3,618 nM, compared to 668 nM in naïvecells. FIG. 21B provides the IC₅₀ curves of the NDC (FA-CDC) (preparedusing drug-linker conjugate precursor 202, of Example 33) in the naïveKB cells (IC₅₀=0.27 nM) and resistant KB cells (IC_(50=0.26) nM),indicating the NDC has a high potency that is uniform across both naïveKB cells and TOP1 inhibitor-resistant KB cells.

Exatecan-Resistant KB Cell Line and Potency Test of FA-CDC

FIG. 22A shows the IC₅₀ curves of exatecan in both naïve and resistantKB cells, which demonstrates the successful development of >8×exatecan-resistant KB cells, were IC50 of exatecan in regular KB cellswas 2 nM, compared with 4 nM in KB cells pretreated 4× with exatecan,and 16.9 nM in KB cells pretreated 7× with exatecan. FIG. 22B shows theIC₅₀ curves of the NDC (FA-CDC) (prepared using the payload-linkerconjugate precursor 202 of Example 33) in both naïve and resistant KBcells (4× or 7× pretreatment), where the IC₅₀ of the FA-CDC was 0.27 nM,0.28 nM, and 0.30 nM, respectively. The results indicated the NDCpossesses high potency uniformly in both the naïve and resistant KBcells.

Example 103: Activity of NDCs in Cancer Cells with Varied FolateReceptor Expression Levels

An assay was conducted to determine the cytotoxicity of exemplary NDCs(FA-CDCs), with varying levels of drug-to-particle ratio, in differentFR-alpha overexpressing cancer cell lines, compared to non-conjugatedexatecan. The NDCs were prepared using the payload-linker conjugate 202provided in Example 33. The NDCs (FA-CDCs) tested had a drug-to-particleratio (DPR) of 43, 20, 8, and 1 (i.e., 43, 20, 8, and 1 exatecan-linkergroups per nanoparticle).

Cancer cells with varied FR alpha expression levels (KB(++++),IGROV-1(++), SK-OV-3(++), HCC827(++), A₅₄₉(−), and BT549(−)) werecultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) forat least one week before the study. Assays for 7-day exposure and 6-hourexposure were both conducted.

Cells were plated in opaque 96-well plates at a density of 3×103 cellsper well (total of 90 μL) and allowed to attach overnight. The followingday, cells were treated with NDC with varied drug-to-particle ratio(DPR) at a concentration ranging from 0-50 nM (0, 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10 μL of 10× stock compounds.

For the 6-hour exposure viability study cells were treated for 6 hoursand washed (3×) with 100 μL PBS. 100 μL of fresh cell medium was thenadded to each well and the plate was incubated for an additional 7 daysat 37° C. before performing the CellTiter-Glo® cytotoxic assay (Promega)according to manufacturer's instructions. The results of the 7-dayexposure assay are presented in FIG. 23 , which demonstrate that the NDCwas highly potent across all cell lines, despite differing levels of FRexpression in the cells.

For the 7-day exposure viability study, the cells were incubated withcompounds for the entire 7-day period, followed by the CellTiter-Glo®cytotoxic assay. Data for half maximal inhibitory concentration (IC₅₀)was plotted using Prism7 software (GraphPad). The results of the 6-hourexposure assay are presented in FIG. 23 , which demonstrate that the NDCwas highly potent across all cell lines, despite differing levels of FRexpression in the cells.

Example 104: Cytotoxicity of NDCs in Human-Derived Pt-Resistant TumorCell Lines

An assay was conducted to establish the cytotoxicity of an exemplary NDC(prepared using the linker-payload conjugate precursor Compound 202,from Example 33) in various patient derived tumor cell lines that arePt-resistant, with comparison to non-conjugated exatecan. Cell lineswere obtained from ovarian cancer, non-small cell lung cancer (NSCLC),breast cancer (both HR+, HER2+; and HR−, HER2+; and triple negativebreast cancer (TNBC)), endometrial cancer, and head and neck (H&N)cancers. The results of the assay are provided in FIG. 24 .

The cytotoxic efficacy was determined by KIYATEC using the KIYA-PREDICT™assay. The FRα immunohistochemistry (ICH) scoring of tumor tissue fromplatinum-resistant ovarian, endometrium, non-small cell lung, breast,triple-negative breast, head & neck cancer patients were conducted byXenoSTART by using the Biocare Medical FRa IHC Assay Kit (cat#BRI4006KAA), following the manufacturer's protocol. A total of 28 PDXmodels from different indications were selected based on the IHC scoresand provided to KIYATEC for the KIYA-PREDICT™ assay. Briefly,cryopreserved PDX tumors were thawed and enzymatically dissociated tosingle cells, and plated into 384-well spheroid microplates (Coming).Flow cytometry was also performed to assess the FRαlevels amongdifferent PDX models. Following the 24 hours of spheroid formation, NDCor controls were added at the designed concentration range and incubatedfor 7 days. After that, the cell viability was measured byCellTiter-Glo® 3D (Promega). The data was analyzed in Microsoft Exceland GraphPad Prism.

Example 104: In Vitro and In Vivo Efficacy of an Exemplary NDC inPediatric Acute Myeloid Leukemia Models

Assays were carried out to establish the in vitro and in vivo efficacyof an exemplary NDC (prepared using the linker-payload conjugateprecursor Compound 202, from Example 33) in folate-receptoralpha-positive pediatric acute myeloid leukemia models.

In Vitro Flow Cytometry Cell Binding Study

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one weekbefore the study. Cell binding studies were performed by incubating5×10⁵ cells (total of 500 μL, 1 million/mL) in cold phosphate-bufferedsaline (PBS) (with 1% of bovine serum albumin (BSA)) with the exemplaryNDC or with anti-FR alpha phycoerythrin (PE)-conjugated antibodies(anti-FR alpha antibody-PE) (concentration: 10 nM) for 60 min at 4° C.(n=3). A non-targeted CDC and isotype antibody-PE were used as negativecontrols for the exemplary NDC and anti-FR alpha antibody-PE,respectively. The cell suspension was then stained with viability kit(LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, Thermo Fisher) for 10-15min. The cells were next centrifuged (2000 revolutions per minute, 5min), washed (2-3 times) using cold PBS (with 1% of BSA) beforeresuspending in PBS (with 1% of BSA). Triplicate samples were analyzedon a LSRFortessa flow cytometer (BD Biosciences) (Cy5 channel, 633nm/647 nm, Live/dead cell stain, 405 nm). Results were processed usingFlowJo and Prism 7 software (GraphPad).

The flow cytometry histograms of the exemplary NDC and anti-FR alphaantibody-PE compared with the respective negative controls (non-targetedNDC or isotype antibody-PE) are shown in FIGS. 25A-25D. The flow studydemonstrates the specific FR alpha targeting capability of the exemplaryNDC to both the IGROV-1 (FR alpha positive human ovarian cancer) and theAML MV4;11 cell lines.

In Vitro CellTiter-Glo® Cytotoxic Assay

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one weekbefore the study. Cells were plated in opaque 96-well plates at adensity of 3×10³ cells per well (total of 90 μL) and allowed to attachovernight. The following day, cells were treated with the exemplary NDCat a concentration ranging from 0-100 nM, by adding 10 μL of 10× stockNDC solution. For the shorter exposure viability study, cells weretreated for 4 hours and washed (3×) with 100 μL PBS. 100 μL of freshcell medium without the NDC was then added to each well and the platewas incubated for an additional 5 days at 37° C. before performing theCellTiter-Glo® cytotoxic assay (Promega) according to manufacturer'sinstructions. Data for half maximal inhibitory concentration (IC₅₀) wasplotted using Prism7 software (GraphPad).

The in vitro specific cytotoxic activity of the exemplary NDC in FRalpha positive human ovarian cancer and MV4;11 AML cell lines isdisplayed in FIGS. 26A-26B. Cells were treated with the exemplary NDC atthe indicated concentrations, incubated at 37° C. for 4 hours, washed,and returned to the incubator for an additional 5 days, beforeperforming the CellTiter-Glo® cytotoxic assay.

CBFA2T₃-GLIS2 Fusion-Positive AML Cell Line-Derived Xenograft Models

In vivo anti-tumor killing activity of the exemplary NDC was assessed incell line-derived xenograft (CDX) models. NOD scid gamma (NSG) mice werefed with folate free chow for 1 week prior to injection with AML celllines. Then 1-5 million fusion-positive cell lines (M07e, WSU-AML) andengineered cells (MV4;11 FOLR+) transduced with Luciferase reporter weretransplanted into the NSG mice via tail-vein injections. Leukemia burdenand response to treatments was monitored using non-invasivebioluminescent imaging (from both the front and the back of the mouse),and flow cytometry analysis of mouse peripheral blood drawn bysubmandibular bleeds was carried out bi-weekly, starting from the firstweek of CDC treatment. Mice were monitored for disease symptoms(including tachypnea, hunchback, persistent weight loss, fatigue, andhind-limb paralysis). Mice from the saline control group (Cohort 1) wereeuthanized due to the high AML burden on Day 44 post-leukemia injection(tissues including blood, bone marrow, thymus, liver, lungs and spleenwere harvested at necropsy and analyzed for the presence of leukemiacells). Mice from the treatment groups (Cohorts 2-4) continued toreceive weekly bioluminescent imaging and bodyweight monitoring. Anillustration of the timeline for mice preparation, treatment, andimaging is provided in FIG. 31 .

All the mice were randomized prior to dosing and weighed to provide thecorrect designed dose based on Table 5 below. Leukemia burden andresponse to treatments was monitored weekly using non-invasivebioluminescent imaging. Bodyweight was measured every other day. Themice were terminated if their weight loss was over 20%.

TABLE 5 Dose design (n = 5 per group) Clinical Dose IV Dose ObservationsMaterial (mg/kg of volume and Study End Cohort Administered Study PhaseExatecan) Regimen (mL/kg) Points 1 Normal saline Vehicle control n/apQ3D × 3 10 Every other day 2 NDC escalation 0.33 Q3D × 6 10 body weight(BW) 3 NDC escalation 0.50 Q3D × 3 10 End point: BW loss 4 NDCescalation 0.65 Q3D × 3 10 >20%

FIG. 27 provides the bodyweight change of AML mice treated with normalsaline and the exemplary NDC at the three dose levels indicated in Table5. The normal saline group (Cohort 1) showed a bodyweight loss within20%, mainly due to the leukemia burden. In the 0.33 mg/kg (Q3D×6) dosegroup (Cohort 2), 4 of 5 mice tolerated the NDC well (<20% loss), andbodyweight was gained after 6 doses; while the remaining mouseshowed >20% bodyweight loss after the 5^(th) dose, and more bodyweightloss after the 6^(th) dose. In the 0.50 mg/kg (Q3D×3) dose group (Cohort3), all 5 mice tolerated the NDC well (<20% loss), and bodyweight wasgained after 3 doses. In the 0.65 mg/kg (Q3D×3) group (Cohort 4), 2 of 5mice tolerated the NDC well (<20% loss), and bodyweight was gained after3 doses; while 3 of 5 mice showed >20% bodyweight loss after the 3^(rd)dose.

FIG. 28 provides the in vivo bioluminescence images (BLI) obtained fromthe AML mice treated with normal saline or the exemplary NDC at eachdose regimen (i.e., Cohorts 1-4 from Table 5). Quantitative in vivobioluminescence imaging analysis of Cohorts 1-4 (i.e., AML mice treatedwith normal saline or the exemplary NDC at each dose regimen outlined inTable 5) is provided in FIG. 29 . In the normal saline group (Cohort 1),the leukemia burden continued to progress, with the average whole-bodyBLI signal increasing >90 fold in 34 days, while a quick anddose-dependent suppression of the leukemia burden was achieved in all 3treatment groups (Cohorts 2-4). The 0.5 mg/kg (Q3D×3) dose group (Cohort3) showed 11-fold less leukemia burden on Day 34 when compared withburden on Day 1 post-leukemia injection. When comparing the 0.33 mg/kg(Q3D×6) dose group (Cohort 2) with the 0.65 mg/kg (Q3D×3) dose group(Cohort 4), 0.33 mg/kg was tolerated better with a slightly betterresponse. Taken together, these data indicate the exemplary NDCsuccessfully suppressed the leukemia burden in the FR alpha positive AMLmice, and showed quick and dose-dependent response.

FIG. 30 provides a graph illustrating the results of bone marrowaspiration of Cohorts 1-4 (i.e., the mice treated with normal saline orthe exemplary NDC at each dose group indicated in Table 5) on Day 42post-leukemia injection. Leukemia was detected in the group of micetreated with normal saline (Cohort 1), while no detectable leukemiaburden could be observed in any of the mice from the treatment groups(Cohorts 2-4).

Example 105. Stability of Linker Derived from Diene

In order to determine the stability of NDCs disclosed herein preparedusing a diene-based functionalization approach, the stability of NDCprepared using a diene based functionalization approach were compared toNDC prepared using an amine-based functionalization approach.

The NDCs were incubated in 0.9% saline, PBS, human plasma (10%), andmouse plasma (10%) at 37° C. in a shaking dry bath for different timeperiods. Prior to analysis, plasma proteins in the samples were removedby precipitation, through addition of an equivalent volume of coldacetonitrile, followed by centrifugation at 10000 rpm in an Eppendorf5425 microcentrifuge. Following centrifugation, the clear supernatantwas transferred from the centrifuge tube into a clear total recoveryHPLC vial. The supernatant free of any visible aggregation was dilutedwith an equivalent volume of deionized water to adjust the sample matrixto match the starting conditions of the HPLC separation and avoid lossof sensitivity. The purity and impurity of each sample was thenquantified by RP-HPLC.

The targeted-NDCs produced using the methods described herein, using adiene-silane precursor, exhibited high stability in mouse and humanplasma, and showed significant stability improvement, relative tocorresponding NDCs produced using an amine-silane precursor (see FIGS.32A and 32B). In the NDCs prepared using the diene-silane precursor,more than 95% of exatecan drugs remained on the NDCs for up to 7 days inmouse and human plasma, obtained by the UV-Vis spectra of the NDC peaksin RP-HPLC chromatograms. Meanwhile, an independent RP-HPLC assaymonitoring free exatecan suggested that the released exatecan was belowdetection limit of RP-HPLC, i.e., 0.02%, and the absence of non-desiredfree drug further demonstrates their high plasma stability. Thetargeted-NDCs also exhibited high storage stability at 4° C. in 0.9%saline. Their purity, size distribution, and hydrodynamic diameter werecharacterized by RP-HPLC, SEC and FCS respectively, and remainedunchanged over 6 months under storage condition. Such high storagestability is another key parameter important for both clinicaltranslation and commercial manufacture.

Example 106. Pharmacokinetics and Toxicology Study

The pharmacokinetics and toxicology of an exemplary NDC were assessed ina rat model and in a dog model. The NDC used in this study was preparedusing the exatecan-linker conjugate precursor compound 202 of Example33. As demonstrated in the above examples, this exemplary NDC is highlystable in plasma and elicits antitumor efficacy in a variety of cellline and PDX-derived tumor models both in vitro and in vivo.

In 15-day repeat dose toxicology and toxicokinetic (TK) studiesperformed in Wistar Han rats and Beagle dogs, the NDC was tolerated atup to 0.87 mg/kg/day in rats and 0.174 mg/kg/day in dogs based uponconjugated exatecan concentration when administered on a QWx3 schedulevia a 1-hour infusion. Observed dose-related toxicities for both specieswere limited to the bone marrow and gastrointestinal tract. These arethe same organs as those observed when free payload (exatecan) wasadministered, suggesting that the delivery of exatecan conjugated to theNDC did not broaden the tissue toxicity profile. Observed toxicitieswere recovered or substantially reduced by the end of a two-weekrecovery period. No drug-related hepatic, renal, pulmonary or oculartoxicities were observed, and there were no drug-related deaths in therepeat dose toxicity study.

TK parameters, estimated in the 15-day GLP study, revealed similarplasma exposure values in males and females for the NDC, total exatecan(conjugated and released) and released exatecan. The NDC exhibited anaverage circulatory half-life ranging from approximately 15 to 20 hoursin rats, and 24 to 29 hours in dogs, with no accumulation of the NDC,total exatecan, or free exatecan observed from day 1 to day 15. Basedupon AUC_(0-last) (hr*ng/mL) released payload levels in the circulationwere less than approximately 0.3% and 0.10% of the total payload levelsin the rat and the dog respectively. No NDC anti-drug antibodies wereinduced in either species. In summary, the NDC has a favorablenonclinical safety/TK profile.

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A nanoparticle-drug conjugate (NDC) comprising: (a) a silicananoparticle that comprises polyethylene glycol (PEG) covalently bondedto the surface of the silica nanoparticle; (b) a targeting ligandselected from the group consisting of folic acid, dihydrofolic acid,tetrahydrofolic acid, and folate receptor binding derivatives of any ofthe foregoing, and wherein the targeting ligand is attached to thesilica nanoparticle directly or indirectly through a spacer group; and(c) a linker-payload conjugate, wherein the payload is a cytotoxicagent; wherein the linker-payload conjugate is attached to the silicananoparticle directly or indirectly through a spacer group; wherein thecytotoxic agent is released upon cleavage of the linker; and wherein thelinker in the linker-payload conjugate is selected from a groupconsisting of protease-cleavable linkers, redox-sensitive linkers, andpH-sensitive linkers.
 2. The NDC of claim 1, wherein the NDC has anaverage diameter between about 1 nm and about 10 nm.
 3. (canceled) 4.The NDC of claim 1, wherein the average silica nanoparticle to payloadratio ranges from 1 to
 80. 5. (canceled)
 6. The NDC of claim 1, whereinthe average nanoparticle to targeting ligand ratio ranges from 1 to 50.7-8. (canceled)
 9. The NDC of claim 1, further comprising a fluorescentcompound covalently encapsulated within the silica nanoparticle.
 10. TheNDC of claim 1, comprising a structure of Formula (NP).

wherein x is an integer of 0 to 20; the silicon atom (Si) is a part ofthe silica nanoparticle; and the

adjacent to the triazole moiety denotes a point of attachment to atargeting ligand or payload-linker conjugate, either directly orindirectly.
 11. The NDC of claim 1, wherein the payload is selected froma group consisting of dihydrofolate reductase inhibitors, thymidylatesynthase inhibitors, and topoisomerase inhibitors.
 12. (canceled) 13.The NDC of claim 1, wherein the payload is a topoisomerase inhibitorselected from a group consisting of SN38, exatecan, and analogs thereof.14. The NDC of claim 1, wherein the NDC comprises a structure of Formula(S-1)

wherein Payload is a cytotoxic agent; Linker is selected from a groupconsisting of protease-cleavable linkers, redox-sensitive linkers, andpH-sensitive linkers, and the silicon atom (Si) is a part of the silicananoparticle.
 15. (canceled)
 16. The NDC of claim 1, wherein the NDCcomprises a structure of Formula (S-2):

wherein the silicon atom (si) is a part of the silica nanoparticle. 17.(canceled)
 18. The NDC of claim 1, wherein the linker-payload conjugatecomprises a structure of Formula (I):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; A is a dipeptide selected fromthe group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val- Lys,Val-Arg, and Val-Ala, or A is a tetrapeptide selected from the groupconsisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar,Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro,Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of a cytotoxic moiety, and when Payload is aseparate molecular entity it contains an amino or hydroxyl group thatprovides the nitrogen or oxygen atom at Z; R¹ and R² in each occurrenceis independently hydrogen, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₁₋₆ alkoxy, or hydroxy; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵ is selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅₋₆ heterocycloalkyl; with the provisothat, when A is a dipeptide, R⁵ is H; R^(a), R^(b) and R^(c) in eachoccurrence is independently hydrogen, or substituted or unsubstitutedC₁₋₆ alkyl; X is absent, —O—, —CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

 is bonded to Z; with the proviso that, when Y is

 X is absent and n is 1; with the proviso that, when Y is

 X is absent and n is 0; with the proviso that, when Y is

 X is absent and n is 0 or 1; with the proviso that, when X is —CO—, Yis absent and n is 0; X₁ and X₂ are independently —CH— or —N—; X₃ is—CH—; X₄ is —CH—; Z is —NR^(c)— or —O—; n is 0 or 1; and q is 1 to 3.19. The NDC of claim 1, wherein the linker-payload conjugate comprises astructure of Formula (II)

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; A is a dipeptide selected fromthe group consisting of Val-Cit, Phe-Lys, Trp- Lys, Asp-Lys, Val- Lys,Val-Arg, and Val-Ala, or A is a tetrapeptide selected from the groupconsisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar,Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro,Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of cytotoxic moiety, and when Payload is aseparate molecular entity it contains an amino or hydroxyl group thatprovides the nitrogen or oxygen atom at Z; R¹ is hydrogen or substitutedor unsubstituted C₁₋₆ alkyl; R³ and R⁴ in each occurrence isindependently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ alkoxy; R⁵ is selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁₋₆ alkyl;substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅₋₆ heterocycloalkyl; with the provisothat, when A is a dipeptide, R⁵ is H; R^(a), R^(b) and R^(c) in eachoccurrence is independently hydrogen or substituted or unsubstitutedC₁₋₆ alkyl; X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is—CH—; Y₁ is

 and Z is —NR^(c)— or —O—.
 20. (canceled)
 21. The NDC of claim 1,wherein the linker-payload conjugate comprises a structure of Formula(IV):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; A is a dipeptide selected fromthe group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val- Lys,Val-Arg, and Val-Ala, or A is a tetrapeptide selected from the groupconsisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar,Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro,Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, Val-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural orunnatural N-alkyl substituted alpha amino acid, Val-Ala-Gly-any naturalor unnatural N-alkyl substituted alpha amino acid, Phe-Lys-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid, andTrp-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid; Payload is a residue of cytotoxic moiety, and when Payload is aseparate molecular entity it contains an amino or hydroxyl group thatprovides the nitrogen or oxygen atom at Z; R^(c) is selected from agroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;and Z is —NR^(c)— or —O—.
 22. (canceled)
 23. The NDC of claim 1, whereinthe linker-payload conjugate comprises a structure of Formula (V)

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R^(a), R^(b) andR^(c) in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; X is absent, —O— or —NR^(a)—; Y is absent,

wherein the carbonyl in

 is bonded to Z; X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄is —CH—; Z is —NR^(c)— or —O—; n is 0 or 1; p is 1 to 3 and q is 1 to 3.24. The NDC of claim 1, wherein the linker-payload conjugate comprises astructure of Formula (VI):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R^(a), R^(b) andR^(c) in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; X₁ and X₂ are independently —CH— or —N—; X₃ is—CH—; X₄ is —CH—; Y₁ is

Z is —NR^(c)— or —O— and p is 1 to
 3. 25. The NDC of claim 1, whereinthe linker-payload conjugate comprises a structure of Formula (VII):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R₁, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R^(c) is selectedfrom a group consisting of hydrogen or substituted or unsubstituted C₁₋₆alkyl; Y is

 wherein the carbonyl in

 is bonded to Z; Z is —NR^(c)— or —O—′ and p is 1 to
 3. 26. (canceled)27. The NDC of claim 1, wherein the linker-payload conjugate comprises astructure of Formula (IX):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R¹, R², R⁵, R⁶ and R⁷ in each occurrence is independentlyhydrogen or substituted or unsubstituted C₁₋₆ alkyl; R^(c) is selectedfrom a group consisting of hydrogen or substituted or unsubstituted C₁₋₆alkyl; Z is —NR^(c)— or —O—; m is 1 to 3; and p is 1 to
 3. 28. The NDCof claim 1, wherein the linker-payload conjugate comprises a structureof Formula (X):

or a salt thereof, wherein,

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group; Payload is a residue ofcytotoxic moiety, and when Payload is a separate molecular entity itcontains an amino or hydroxyl group that provides the nitrogen or oxygenatom at Z; R⁵, R⁶ and R⁷ in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; R³ and R⁴ in each occurrence isindependently hydrogen, halo, substituted or unsubstituted C₁₋₆ alkyl,or substituted or unsubstituted C₁₋₆ alkoxy; R^(c) is selected from agroup consisting of hydrogen or substituted or unsubstituted C₁₋₆ alkyl;X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is—NR^(c)— or —O—; and p is 1 to
 3. 29-32. (canceled)
 33. A method oftreating cancer, comprising administering to a subject in need thereofan effective amount of a nanoparticle drug conjugate (NDC), wherein theNDC comprises: (a) a silica nanoparticle that comprises polyethyleneglycol (PEG) covalently bonded to the surface of the silicananoparticle; (b) a targeting ligand that binds to a folate receptor:and (c) a linker-payload conjugate, wherein the linker-payload conjugateis attached to the silica nanoparticle directly or indirectly through aspacer group: and wherein the payload is released upon cleavage of thelinker. 34-35. (canceled)
 36. The method of claim 33, wherein the NDC isadministered to the subject intravenously.
 37. The method of claim 33,wherein the subject has a cancer selected from the group consisting ofovarian cancer, endometrial cancer, fallopian tube cancer, cervicalcancer breast cancer, lung cancer, mesothelioma, uterine cancer,gastrointestinal cancer, pancreatic cancer, bladder cancer, kidneycancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia and chronic myelogenous leukemia (CML). 38-46. (canceled)
 47. Apharmaceutical composition comprising an NDC of claim 1, and apharmaceutically acceptable excipient. 48-50. (canceled)
 51. Alinker-drug conjugate of any one of Formulae (I)-(XII) or I-B-XII-B).52. The linker-drug conjugate of claim 51, comprising a carrier particleselected from the group consisting of a nanoparticle, a liposome, ananogel, a nanoring, a nanocage, a microsphere, an antibody, anantigen-binding portion or fragment of an antibody, a minibody, and ananobody.
 53. A method of treating cancer, comprising administering to asubject in need thereof an effective amount of a linker-drug conjugateof claim
 51. 54-55. (canceled)
 56. A linker of any one of Formulae(I-A)-(X-A).